[Objective] The research aimed to construct the fusion protein expression vector of α-galactosidase-EGFP （enhanced green fluorescent protein） in cucumber controlled by CaMV35S promoter.[Method] CaMV35S promoter seq...[Objective] The research aimed to construct the fusion protein expression vector of α-galactosidase-EGFP （enhanced green fluorescent protein） in cucumber controlled by CaMV35S promoter.[Method] CaMV35S promoter sequence and the coding region of EGFP were amplified by polymerase chain reactions （PCR） with vector pCambia 1303 as the template.Using reverse transcript PCR technology,with total RNAs of cucumber as template,the coding region of acid α-galactosidase Ⅰ in cucumber was amplified.The above three fragments were inserted into the multiple cloning sites of expression vector pCambia 1381c.The fusion expression vector of α-galactosidase-EGFP located at the C-terminal of the target genes was constructed.[Result] After enzyme digestion and sequencing,the fusion expression of α-galactosidase-EGFP in cucumber was constructed successfully.[Conclusion] The research laid the experimental basis for further study on the subcellular localization of α-galactosidase in cucumber.展开更多
The work is intended to achieve optimum culture conditions of α-galactosidase production by a mutant strain ,Aspergillusfoetidus ZU-GI in solid-state fermentation （SSF）. Certain fermentation parameters involving mo...The work is intended to achieve optimum culture conditions of α-galactosidase production by a mutant strain ,Aspergillusfoetidus ZU-GI in solid-state fermentation （SSF）. Certain fermentation parameters involving moisture content, incubation temperature, cultivation period of seed, inoculum volume, initial pH value, layers of pledget, load size of medium and period of cultivation were investigated separately. The optimal cultivating conditions of α-galactosidase production in SSF were 60% initial moisture of medium, 28 ℃ incubation temperature, 18^h cultivation period of seed, 10% inoculum volume, 5.0-6.0 initial pH of medium, 6 layers of pledget and 10 g dry matter loadage. Under the optimized cultivation conditions, the maximum α-galactosidase production was 2037.51 U/g dry matter near the 144th hour of fermentation.展开更多
Construction of a food-grade expression vector for application to lactic acid bacteria（LAB） is of importance for dairy fermentation system. An α-galactosidase（aga） gene encoding an enzyme degrading melibiose was ...Construction of a food-grade expression vector for application to lactic acid bacteria（LAB） is of importance for dairy fermentation system. An α-galactosidase（aga） gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid p RAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36 e to substitute the p rimary antibiotic selectable marker of pMG36 e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long α-amylase（amy） gene from Ba cillus li cheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. Th e selection efficiency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU ？g-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putati ve molecular weight of α-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of α-amylase by expressing of amy gene of pMG36-aga-amy.展开更多
Background Human group O red blood cells have great benefit in specialized transfusion areas such as armed conflict and natural calamity. The group B antigen differs structurally from group O antigen only by the addit...Background Human group O red blood cells have great benefit in specialized transfusion areas such as armed conflict and natural calamity. The group B antigen differs structurally from group O antigen only by the addition of one terminal α-linked galactose residue. In this study we aimed to remove the terminal galactose from group B red blood cell to get group O red blood cell. Methods α-galactosidase cDNA was cloned by RT-PCR from Catimor coffee beans grown on Hainan Island of China. The vector for α-galactosidase cDNA expression was constructed and transferred into Pichia pastoris cells by electroporation. The transgenic cells were cloned by fermentation and the recombinant α-galactosidase was purified by ion exchange chromatography. After studying the biochemical characters of α-galactosidase, we have used it in converting human erythrocytes from group B to group O. Results The purity of recombinant α-galactosidase was higher than 96%, which was thought to be suitable for the use of blood conversion. Enzymatically converted human group O red blood cells （ECHORBC） exhibited membrane integrity, metabolic integrity, normal cell deformation and morphology. There were no coagulation between ECHORBC and any group of human blood. The ECHORBC will keep normal structure and function for a period of 21 days at 4℃ in monoammoniumphosphate nutrient solution. Experiments with Rhesus monkeys and gibbons showed that transfusion of enzymatically converted erythrocytes was safe. Conclusion ECHORBC can be easily obtained from group B red blood cell by α-galactosidase digestion. This study suggests that ECHORBC could be transfused to patients safely and efficiently.展开更多
Objective To construct four recombinant Lactococcus lactis strains exhibiting high β-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion....Objective To construct four recombinant Lactococcus lactis strains exhibiting high β-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion. Methods The gene fragments encoding β-galactosidase from two strains of Loctobacillus bulgaricus, wch9901 isolated from yogurt and 1.1480 purchased from the Chinese Academy of Sciences, were amplified and inserted into lactococcal expression vector pMG36e. For fusion expression, the open reading frame of the β-galactosidase gene was amplified, while for non-fusion expression, the open reading frame of the β-galactosidase gene was amplified with its native Shine-Dalgarno sequence upstream. The start codon of the β-galactosidase gene partially overlapped with the stop codon of vector origin open reading frame. Then, the recombinant plasmids were transformed into Escherichia coli DH5α and Lactococcus lactis subsp, lactis MG1363 and confirmed by determining β-galactosidase activities. Results The non-fusion expression plasmids showed a significantly higher β-galactosidase activity in transformed strains than the fusion expression plasmids. The highest enzyme activity was observed in Lactococcus lactis transformed with the non-fusion expression plasmids which were inserted into the β-galactosidase gene from Lactobacillus bulgaricus wch9901. The β-galactosidase activity was 2.75 times as high as that of the native counterpart. In addition, β-galactosidase expressed by recombinant plasmids in Lactococcus lactis could be secreted into the culture medium. The highest secretion rate （27.1%） was observed when the culture medium contained 20 g/L of lactose. Conclusion Different properties of the native bacteria may have some effects on the protein expression of recombinant plasmids. Non-fusion expression shows a higher enzyme activity in host bacteria. There may be a host-related weak secretion signal peptide gene within the structure gene of Lb. bulgaricus β-galactosidase, and its translation product may introduce the enzyme secretion out of cells in special hosts.展开更多
Objective This study is to examine the secretion effects of β-galactosidase in Lactococcus lactis.Methods The usp45 and β-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant pl...Objective This study is to examine the secretion effects of β-galactosidase in Lactococcus lactis.Methods The usp45 and β-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant plasmid pMG36e-usp-lacZ.This recombinant plasmid was transformed into both Escherichia coli DH5α and L.lactis MG1363.The enzyme activity,gene sequencing,SDS-PAGE and hereditary stability were assessed and studied.Results The lacZ gene inserted into plasmids pMG36e-usp-lacZ was 99.37% similar to the GenBank sequence,and SDS-PAGE revealed an evident idio-strap at 116 KDa between L.lactis MG1363/pMG36eusp-lacZ in both supernatant and cell samples.β-Galactosidase activity measured 0.225 U/mL in L.lactis pMG36e-usp-lacZ transformants,and its secretion rate was 10%.The plasmid pMG36e-usp-lacZ appeared more stable in MG1363.Conclusion The authors concluded that these new recombinant bacteria well expressed and secreted β-galactosidase,indicating that the β-galactosidase expression system was successfully constructed,and this might provide a new solution for management of lactose intolerance specifically and promote the use of gene-modified organisms as part of the food-grade plasmid in general.展开更多
The performance of gold and polystyrene nanoparticles was investigated using quartz crystal microbalance (QCM) as sensor platform;β-Galactosidase antibody with corresponding antigen was utilized for the immunoreactio...The performance of gold and polystyrene nanoparticles was investigated using quartz crystal microbalance (QCM) as sensor platform;β-Galactosidase antibody with corresponding antigen was utilized for the immunoreaction. The development of the immunosensor included: 1) formation of self assembled monolayers on quartz crystals;2a) immobilization of p-aminothiophenol functionalized gold nanoparticles on carboxyl-terminated self assembled monolayer, or 2b) immobilization of polystyrene nanoparticles on crystals modified with p-aminothiophenol self assembled monolayer;3) attachment of monoclonal anti β-Gal on nanoparticles;and 4) detection of target analyte. The nanoparticles used were synthesized in house and characterized by transmission electron microscopy and infrared spectroscopy. The results revealed that antibodies were strongly attached to functionalized gold nanoparticles;the weaker immobilization of antibodies to polystyrene nanoparticles provoked their detachment during antigen detection. When cross reactivity of polystyrene nanoparticles was checked using a different antigen (Brucella), displacement of antibody was not recorded, demonstrating specificity of the reaction. To the best of our knowledge this is the first direct comparison between behaviors of biosensors developed with two commonly used nanoparticles. The results showed that both nanoparticles produced biosensors capable to detect β-Gal. Nevertheless biosensors developed using polystyrene nanoparticles are simpler, cheaper and more eco-friendly than those developed using gold nanoparticles.展开更多
β-galactosidase was extracted from apricots (Prunus armeniaca kaisa) and characterized biochemically. Three isoenzymes (β-gal I, β-gal II and β-gal III) were obtained by salt fractionation and ionexchange and Seph...β-galactosidase was extracted from apricots (Prunus armeniaca kaisa) and characterized biochemically. Three isoenzymes (β-gal I, β-gal II and β-gal III) were obtained by salt fractionation and ionexchange and Sephadex G-100 column chromatography. β-galactosidase II showed a high ability to hy-drolyze the substrate p-nitrophenyl β-D-galactopyranoside than that of β-galactosidase I and III. The individual peaks showed charge homogeneity as revealed by single band on polyacrylamide gel. The molecular weight of β-gal I, β-gal II and β-gal III as determined by gel filtration was found to be 44.15, 34.70 and 23.71 KDa respectively. The optimum pH for the activity different isozymes was found between 4 and 6. The isoenzymes were determined to be thermally stable upto 40?C. The Km value for β-gal I was 1.85 mM which was higher than that of β-gal II (Km = 1.7), and β-gal III (Km = 1.19). The Vmax value for β-gal I, β-gal II and β-gal III was found to be 0.52, 0.70 and 0.38 μmole/min respectively.展开更多
The enzyme β-galactosidase （lactase; EC 3.2.1.23） is a commercially important enzyme due to its various applications in dairy and food industries, which are based on the β-galactosidase-catalysed hydrolysis of lac...The enzyme β-galactosidase （lactase; EC 3.2.1.23） is a commercially important enzyme due to its various applications in dairy and food industries, which are based on the β-galactosidase-catalysed hydrolysis of lactose into glucose and galactose. The objectives of this work were to identify novel and attractive sources of this industrially relevant enzyme, and to study the effect of selected growth parameters （carbon source, lactose concentration, nitrogen source, peptone concentration, initial pH and temperature） on the formation of β-galactosidase. Based on a screening of isolates from Tha Pai hot spring, Mae Hong Son Province, Thailand, strain BI.1 was selected for further studies. Strain BI.1 is a Gram-positive, rod-shaped, catalase-positive bacterium that forms endospores. Based on the sequence of the 16S rDNA determined, this isolate is most closely related to Anoxybacillus sp. and Bacillus sp., and hence the strain is designated as Bacillus sp. B 1. I.β-Galactosidase was produced by this strain with lactose and peptone as carbon and nitrogen sources, respectively. Optimal enzyme production occurred at an initial culture pH of 8.5 and at 45 ℃. Under these optimum culture conditions, maximal volumetric and specific β-galactosidase activity of 0.478 U mL^-1 and 0.338 U mg^-1 protein, respectively, were obtained after 13 h of cultivation in a medium contain 2.5% lactose, 2.0% peptone, 0.3% K2HPO4, 0.1% KH2PO4 and 0.05% MgSOa·7H2O.展开更多
BACKGROUND The pathophysiology of Fabry disease(FD)-induced progressive vital organ damage is irreversible.Disease progression can be delayed using enzyme replacement therapy(ERT).In patients with classic FD,sporadic ...BACKGROUND The pathophysiology of Fabry disease(FD)-induced progressive vital organ damage is irreversible.Disease progression can be delayed using enzyme replacement therapy(ERT).In patients with classic FD,sporadic accumulation of globotriaosylceramide(GL-3)in the heart and kidney begins in utero;however,until childhood,GL-3 accumulation is mild and reversible and can be restored by ERT.The current consensus is that ERT initiation during early childhood is paramount.Nonetheless,complete recovery of organs in patients with advanced FD is challenging.CASE SUMMARY Two related male patients,an uncle(patient 1)and nephew(patient 2),presented with classic FD.Both patients were treated by us.Patient 1 was in his 50s,and ERT was initiated following end-organ damage;this was subsequently ineffective.He developed cerebral infarction and died of sudden cardiac arrest.Patient 2 was in his mid-30s,and ERT was initiated when the patient was diagnosed with FD,during which the damage to vital organs was not overtly apparent.Although he had left ventricular hypertrophy at the beginning of this treatment,the degree of hypertrophy progression was limited to a minimal range after>18 years of ERT.CONCLUSION We obtained discouraging ERT outcomes for older patients but encouraging outcomes for younger adults with classic FD.展开更多
Fabry Disease (FD) is a rare lysosomal storage disorder characterized by α-galactosidase A (α-Gal A) enzyme deficiency, resulting in glycosphingolipid accumulation. Its clinical spectrum ranges from severe classical...Fabry Disease (FD) is a rare lysosomal storage disorder characterized by α-galactosidase A (α-Gal A) enzyme deficiency, resulting in glycosphingolipid accumulation. Its clinical spectrum ranges from severe classical to milder nonclassical or late-onset phenotypes. Renal involvement, termed Fabry Nephropathy (FN), can vary from mild proteinuria to kidney failure. FN diagnosis, especially in nonclassical cases with a genetic Variant of Unknown Significance (VUS) in the GLA gene, poses challenges. Measurement of plasma lyso-Gb3 levels is gaining importance in FN diagnosis, while renal biopsy with electron microscopy remains the gold standard in equivocal cases. Treatment options include Enzyme Replacement Therapy (ERT) and chaperone therapy, demanding careful candidate selection due to high treatment costs. Research has predominantly focused on classical FD, revealing modest treatment benefits. However, evidence for treating patients, especially females, with milder nonclassical or late-onset phenotypes is scarce, emphasizing the necessity for placebo-controlled clinical trials in these subgroups. Meanwhile, participation in global FD registries can improve our understanding of disease management. Case Presentation: A woman in her late sixties presented with moderate chronic kidney disease, mild proteinuria, and microscopic hematuria. Her family history included a prevalence of renal, cardiac and cerebrovascular diseases. Kidney biopsy revealed characteristic myelin figures and zebra bodies in podocytes, strongly suggestive of FN. Genetic analysis identified a VUS in the GLA gene (c.655A > C, p.Ile219Leu), introducing diagnostic uncertainty. Further investigations revealed severe cardiac involvement. Considering the recurring difficulty presented by the finding of a VUS in the GLA gene during FN assessments, along with the uncertainty regarding the need for treatment in nonclassical or late-onset FD phenotypes, especially in women, this case becomes a central focus for a thorough review of the literature. This review aims to propose a practical algorithm that integrates clinical, biochemical, and genetic markers for FN screening and diagnosis. Additionally, it explores treatment benefits in nonclassical or late-onset FD phenotypes, with a focus on female patients.展开更多
Objective:Organoids are a powerful tool with broad application prospects in biomedicine.Notably,they provide alternatives to animal models for testing potential drugs before clinical trials.However,the number of passa...Objective:Organoids are a powerful tool with broad application prospects in biomedicine.Notably,they provide alternatives to animal models for testing potential drugs before clinical trials.However,the number of passages for which organoids maintain cellular vitality ex vivo remains unclear.Methods:Herein,we constructed 55 gastric organoids from 35 individuals,serially passaged the organoids,and captured microscopic images for phenotypic evaluation.Senescence-associatedβ-galactosidase(SA-β-Gal),cell diameter in suspension,and gene expression reflecting cell cycle regulation were examined.The YOLOv3 object detection algorithm integrated with a convolutional block attention module(CBAM)was used to evaluate organoid vitality.Results:SA-β-Gal staining intensity;single-cell diameter;and expression of p15,p16,p21,CCNA2,CCNE2,and LMNB1 reflected the progression of aging in organoids during passaging.The CBAM-YOLOv3 algorithm precisely evaluated aging organoids on the basis of organoid average diameter,organoid number,and number×diameter,and the findings positively correlated with SA-β-Gal staining and single-cell diameter.Organoids derived from normal gastric mucosa had limited passaging ability(passages 1–5),before aging,whereas tumor organoids showed unlimited passaging potential for more than 45 passages(511 days)without showing clear senescence.Conclusions:Given the lack of indicators for evaluating organoid growth status,we established a reliable approach for integrated analysis of phenotypic parameters that uses an artificial intelligence algorithm to indicate organoid vitality.This method enables precise evaluation of organoid status in biomedical studies and monitoring of living biobanks.展开更多
Aging-related diseases are gradually becoming a major problem with the rapid development of aged population in human society.Although many fluorescent probes have been employed to diagnosis senescence via imaging sene...Aging-related diseases are gradually becoming a major problem with the rapid development of aged population in human society.Although many fluorescent probes have been employed to diagnosis senescence via imaging senescence-associatedβ-galactosidase(SA-β-Gal),which is proved to be closely associated with senescent cells,the similar catalytic effectiveness of enzymatic reaction of ovarian cancer-associatedβ-Gal(OA-β-Gal)will interfere with imaging accuracy.Herein,a near-infrared(NIR)hemicyanine based fluorescent probe HCyXA-βGal was designed for light-up imaging of live cells containingβ-Gal.With the organelle-targeting morpholinyl and positive charge moieties,HCyxA-βGal was successfully applicated to image the difference of enzymatic location in senescent cells and ovarian cancer cells.Furthermore,inspired by the fast response performance,fast and precise imaging of the two cell lines was realized via covering another dimension of fluorescence signal:time-dependent intensity.展开更多
To investigate the relationships among β-mannanase, β-mannosidase and a-galactosidase required for degrading galactomannan in cell wall during and following rice seed germination, the activities of the three enzymes...To investigate the relationships among β-mannanase, β-mannosidase and a-galactosidase required for degrading galactomannan in cell wall during and following rice seed germination, the activities of the three enzymes and the effects of ABA and GA3 on them were surveyed. The activities of β-mannosidase and a-galactosidase presented in dry and pre-germinated rice seeds, and increased slowly during and following germination. However, the activity of β-mannanase was detected only after germination. GA3 could promote the activities of β-mannanase and a-galactosidase. ABA had little effect on the activities of β-mannosidase and α-galactosidase, but it could seriously inhibit the activity of β-mannanase.展开更多
A retroviral vector pINC-lacZ containing an E coli β-galactosidase(β-gal)gene was constructed and introduced into the MESPU-13 cells by electroporation.Four G418-resistant colonies were obtained from 1×107,elec...A retroviral vector pINC-lacZ containing an E coli β-galactosidase(β-gal)gene was constructed and introduced into the MESPU-13 cells by electroporation.Four G418-resistant colonies were obtained from 1×107,electroporated MESPU-13 cells. Histochemical staining for β-gal activity showed that lacZ gene was expressed in at least three of the four colonies.Southern analysis proved that one copy of foreign gene was integrated in the chromosome of one of the transformed lines(MC15).These results showed that the expression of lacZ gene driven by SiCMVIE promoter can be detected in the transformed MESPU-13 cells.展开更多
β-galactosidases (EC 3.2.1.23) constitute a widespread family of glycosyl hydrolases in plants and are thought to be involved in metabolism of cell wall polysaccharides. A cDNA of the cotton (Gossypium hirsutum) β-g...β-galactosidases (EC 3.2.1.23) constitute a widespread family of glycosyl hydrolases in plants and are thought to be involved in metabolism of cell wall polysaccharides. A cDNA of the cotton (Gossypium hirsutum) β-galactosidase gene, designated GhGal1, has previously been identified and its transcripts are highly abundant at the elongation stage of the cotton fiber. To examine the temporal and spatial control of GhGal1 expression, a transcriptional fusion of the GhGal1 promoter region (1770 bp) with the β-glucuronidase (GUS) reporter gene was introduced into tobacco plants by the Agrobacterium infection method. The resulting transgenic plants showed higher GUS activity of fruit in the transgenic plants than that in the negative and positive controls. Histochemical localization of GUS activity demonstrated that the expression of the GUS gene could be found in the meristem zones of roots, cotyledons, vascular tissues, fruit and trichomes in transgenic tobacco plants. Additionally, se-quence analysis of the regulatory region also revealed several conserved motifs among which some were shared with previously reported fruit/seed-specific elements and the others were related with trichome expression. These results indicated the temporal and spatial expression characterization of the GhGal1 promoter in transgenic tobacco plants and provided an important insight into the roles of GhGal1 in cotton fiber development.展开更多
基金Supported by National Basic Research Program of China( 2009CB119000)National Natural Science Foundation(30871721)~~
文摘[Objective] The research aimed to construct the fusion protein expression vector of α-galactosidase-EGFP （enhanced green fluorescent protein） in cucumber controlled by CaMV35S promoter.[Method] CaMV35S promoter sequence and the coding region of EGFP were amplified by polymerase chain reactions （PCR） with vector pCambia 1303 as the template.Using reverse transcript PCR technology,with total RNAs of cucumber as template,the coding region of acid α-galactosidase Ⅰ in cucumber was amplified.The above three fragments were inserted into the multiple cloning sites of expression vector pCambia 1381c.The fusion expression vector of α-galactosidase-EGFP located at the C-terminal of the target genes was constructed.[Result] After enzyme digestion and sequencing,the fusion expression of α-galactosidase-EGFP in cucumber was constructed successfully.[Conclusion] The research laid the experimental basis for further study on the subcellular localization of α-galactosidase in cucumber.
文摘The work is intended to achieve optimum culture conditions of α-galactosidase production by a mutant strain ,Aspergillusfoetidus ZU-GI in solid-state fermentation （SSF）. Certain fermentation parameters involving moisture content, incubation temperature, cultivation period of seed, inoculum volume, initial pH value, layers of pledget, load size of medium and period of cultivation were investigated separately. The optimal cultivating conditions of α-galactosidase production in SSF were 60% initial moisture of medium, 28 ℃ incubation temperature, 18^h cultivation period of seed, 10% inoculum volume, 5.0-6.0 initial pH of medium, 6 layers of pledget and 10 g dry matter loadage. Under the optimized cultivation conditions, the maximum α-galactosidase production was 2037.51 U/g dry matter near the 144th hour of fermentation.
基金supported by the National High-Tech R&D Program of China (863 Program, 2006AA10Z317)
文摘Construction of a food-grade expression vector for application to lactic acid bacteria（LAB） is of importance for dairy fermentation system. An α-galactosidase（aga） gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid p RAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36 e to substitute the p rimary antibiotic selectable marker of pMG36 e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long α-amylase（amy） gene from Ba cillus li cheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. Th e selection efficiency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU ？g-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putati ve molecular weight of α-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of α-amylase by expressing of amy gene of pMG36-aga-amy.
基金the guants from the "973" project (No.2002CB713804)the Military Key Project(No. 2000252910).
文摘Background Human group O red blood cells have great benefit in specialized transfusion areas such as armed conflict and natural calamity. The group B antigen differs structurally from group O antigen only by the addition of one terminal α-linked galactose residue. In this study we aimed to remove the terminal galactose from group B red blood cell to get group O red blood cell. Methods α-galactosidase cDNA was cloned by RT-PCR from Catimor coffee beans grown on Hainan Island of China. The vector for α-galactosidase cDNA expression was constructed and transferred into Pichia pastoris cells by electroporation. The transgenic cells were cloned by fermentation and the recombinant α-galactosidase was purified by ion exchange chromatography. After studying the biochemical characters of α-galactosidase, we have used it in converting human erythrocytes from group B to group O. Results The purity of recombinant α-galactosidase was higher than 96%, which was thought to be suitable for the use of blood conversion. Enzymatically converted human group O red blood cells （ECHORBC） exhibited membrane integrity, metabolic integrity, normal cell deformation and morphology. There were no coagulation between ECHORBC and any group of human blood. The ECHORBC will keep normal structure and function for a period of 21 days at 4℃ in monoammoniumphosphate nutrient solution. Experiments with Rhesus monkeys and gibbons showed that transfusion of enzymatically converted erythrocytes was safe. Conclusion ECHORBC can be easily obtained from group B red blood cell by α-galactosidase digestion. This study suggests that ECHORBC could be transfused to patients safely and efficiently.
基金a scientific research grant from Health Bureau of Sichuan Province (No. F0201)
文摘Objective To construct four recombinant Lactococcus lactis strains exhibiting high β-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion. Methods The gene fragments encoding β-galactosidase from two strains of Loctobacillus bulgaricus, wch9901 isolated from yogurt and 1.1480 purchased from the Chinese Academy of Sciences, were amplified and inserted into lactococcal expression vector pMG36e. For fusion expression, the open reading frame of the β-galactosidase gene was amplified, while for non-fusion expression, the open reading frame of the β-galactosidase gene was amplified with its native Shine-Dalgarno sequence upstream. The start codon of the β-galactosidase gene partially overlapped with the stop codon of vector origin open reading frame. Then, the recombinant plasmids were transformed into Escherichia coli DH5α and Lactococcus lactis subsp, lactis MG1363 and confirmed by determining β-galactosidase activities. Results The non-fusion expression plasmids showed a significantly higher β-galactosidase activity in transformed strains than the fusion expression plasmids. The highest enzyme activity was observed in Lactococcus lactis transformed with the non-fusion expression plasmids which were inserted into the β-galactosidase gene from Lactobacillus bulgaricus wch9901. The β-galactosidase activity was 2.75 times as high as that of the native counterpart. In addition, β-galactosidase expressed by recombinant plasmids in Lactococcus lactis could be secreted into the culture medium. The highest secretion rate （27.1%） was observed when the culture medium contained 20 g/L of lactose. Conclusion Different properties of the native bacteria may have some effects on the protein expression of recombinant plasmids. Non-fusion expression shows a higher enzyme activity in host bacteria. There may be a host-related weak secretion signal peptide gene within the structure gene of Lb. bulgaricus β-galactosidase, and its translation product may introduce the enzyme secretion out of cells in special hosts.
基金supported by the National Science Foundation of China (NO. 30800910)
文摘Objective This study is to examine the secretion effects of β-galactosidase in Lactococcus lactis.Methods The usp45 and β-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant plasmid pMG36e-usp-lacZ.This recombinant plasmid was transformed into both Escherichia coli DH5α and L.lactis MG1363.The enzyme activity,gene sequencing,SDS-PAGE and hereditary stability were assessed and studied.Results The lacZ gene inserted into plasmids pMG36e-usp-lacZ was 99.37% similar to the GenBank sequence,and SDS-PAGE revealed an evident idio-strap at 116 KDa between L.lactis MG1363/pMG36eusp-lacZ in both supernatant and cell samples.β-Galactosidase activity measured 0.225 U/mL in L.lactis pMG36e-usp-lacZ transformants,and its secretion rate was 10%.The plasmid pMG36e-usp-lacZ appeared more stable in MG1363.Conclusion The authors concluded that these new recombinant bacteria well expressed and secreted β-galactosidase,indicating that the β-galactosidase expression system was successfully constructed,and this might provide a new solution for management of lactose intolerance specifically and promote the use of gene-modified organisms as part of the food-grade plasmid in general.
文摘The performance of gold and polystyrene nanoparticles was investigated using quartz crystal microbalance (QCM) as sensor platform;β-Galactosidase antibody with corresponding antigen was utilized for the immunoreaction. The development of the immunosensor included: 1) formation of self assembled monolayers on quartz crystals;2a) immobilization of p-aminothiophenol functionalized gold nanoparticles on carboxyl-terminated self assembled monolayer, or 2b) immobilization of polystyrene nanoparticles on crystals modified with p-aminothiophenol self assembled monolayer;3) attachment of monoclonal anti β-Gal on nanoparticles;and 4) detection of target analyte. The nanoparticles used were synthesized in house and characterized by transmission electron microscopy and infrared spectroscopy. The results revealed that antibodies were strongly attached to functionalized gold nanoparticles;the weaker immobilization of antibodies to polystyrene nanoparticles provoked their detachment during antigen detection. When cross reactivity of polystyrene nanoparticles was checked using a different antigen (Brucella), displacement of antibody was not recorded, demonstrating specificity of the reaction. To the best of our knowledge this is the first direct comparison between behaviors of biosensors developed with two commonly used nanoparticles. The results showed that both nanoparticles produced biosensors capable to detect β-Gal. Nevertheless biosensors developed using polystyrene nanoparticles are simpler, cheaper and more eco-friendly than those developed using gold nanoparticles.
文摘β-galactosidase was extracted from apricots (Prunus armeniaca kaisa) and characterized biochemically. Three isoenzymes (β-gal I, β-gal II and β-gal III) were obtained by salt fractionation and ionexchange and Sephadex G-100 column chromatography. β-galactosidase II showed a high ability to hy-drolyze the substrate p-nitrophenyl β-D-galactopyranoside than that of β-galactosidase I and III. The individual peaks showed charge homogeneity as revealed by single band on polyacrylamide gel. The molecular weight of β-gal I, β-gal II and β-gal III as determined by gel filtration was found to be 44.15, 34.70 and 23.71 KDa respectively. The optimum pH for the activity different isozymes was found between 4 and 6. The isoenzymes were determined to be thermally stable upto 40?C. The Km value for β-gal I was 1.85 mM which was higher than that of β-gal II (Km = 1.7), and β-gal III (Km = 1.19). The Vmax value for β-gal I, β-gal II and β-gal III was found to be 0.52, 0.70 and 0.38 μmole/min respectively.
文摘The enzyme β-galactosidase （lactase; EC 3.2.1.23） is a commercially important enzyme due to its various applications in dairy and food industries, which are based on the β-galactosidase-catalysed hydrolysis of lactose into glucose and galactose. The objectives of this work were to identify novel and attractive sources of this industrially relevant enzyme, and to study the effect of selected growth parameters （carbon source, lactose concentration, nitrogen source, peptone concentration, initial pH and temperature） on the formation of β-galactosidase. Based on a screening of isolates from Tha Pai hot spring, Mae Hong Son Province, Thailand, strain BI.1 was selected for further studies. Strain BI.1 is a Gram-positive, rod-shaped, catalase-positive bacterium that forms endospores. Based on the sequence of the 16S rDNA determined, this isolate is most closely related to Anoxybacillus sp. and Bacillus sp., and hence the strain is designated as Bacillus sp. B 1. I.β-Galactosidase was produced by this strain with lactose and peptone as carbon and nitrogen sources, respectively. Optimal enzyme production occurred at an initial culture pH of 8.5 and at 45 ℃. Under these optimum culture conditions, maximal volumetric and specific β-galactosidase activity of 0.478 U mL^-1 and 0.338 U mg^-1 protein, respectively, were obtained after 13 h of cultivation in a medium contain 2.5% lactose, 2.0% peptone, 0.3% K2HPO4, 0.1% KH2PO4 and 0.05% MgSOa·7H2O.
基金Supported by the Red Cross Hospital Research and Training Fund,Fukushima R.C.Hosp.No.57.
文摘BACKGROUND The pathophysiology of Fabry disease(FD)-induced progressive vital organ damage is irreversible.Disease progression can be delayed using enzyme replacement therapy(ERT).In patients with classic FD,sporadic accumulation of globotriaosylceramide(GL-3)in the heart and kidney begins in utero;however,until childhood,GL-3 accumulation is mild and reversible and can be restored by ERT.The current consensus is that ERT initiation during early childhood is paramount.Nonetheless,complete recovery of organs in patients with advanced FD is challenging.CASE SUMMARY Two related male patients,an uncle(patient 1)and nephew(patient 2),presented with classic FD.Both patients were treated by us.Patient 1 was in his 50s,and ERT was initiated following end-organ damage;this was subsequently ineffective.He developed cerebral infarction and died of sudden cardiac arrest.Patient 2 was in his mid-30s,and ERT was initiated when the patient was diagnosed with FD,during which the damage to vital organs was not overtly apparent.Although he had left ventricular hypertrophy at the beginning of this treatment,the degree of hypertrophy progression was limited to a minimal range after>18 years of ERT.CONCLUSION We obtained discouraging ERT outcomes for older patients but encouraging outcomes for younger adults with classic FD.
文摘Fabry Disease (FD) is a rare lysosomal storage disorder characterized by α-galactosidase A (α-Gal A) enzyme deficiency, resulting in glycosphingolipid accumulation. Its clinical spectrum ranges from severe classical to milder nonclassical or late-onset phenotypes. Renal involvement, termed Fabry Nephropathy (FN), can vary from mild proteinuria to kidney failure. FN diagnosis, especially in nonclassical cases with a genetic Variant of Unknown Significance (VUS) in the GLA gene, poses challenges. Measurement of plasma lyso-Gb3 levels is gaining importance in FN diagnosis, while renal biopsy with electron microscopy remains the gold standard in equivocal cases. Treatment options include Enzyme Replacement Therapy (ERT) and chaperone therapy, demanding careful candidate selection due to high treatment costs. Research has predominantly focused on classical FD, revealing modest treatment benefits. However, evidence for treating patients, especially females, with milder nonclassical or late-onset phenotypes is scarce, emphasizing the necessity for placebo-controlled clinical trials in these subgroups. Meanwhile, participation in global FD registries can improve our understanding of disease management. Case Presentation: A woman in her late sixties presented with moderate chronic kidney disease, mild proteinuria, and microscopic hematuria. Her family history included a prevalence of renal, cardiac and cerebrovascular diseases. Kidney biopsy revealed characteristic myelin figures and zebra bodies in podocytes, strongly suggestive of FN. Genetic analysis identified a VUS in the GLA gene (c.655A > C, p.Ile219Leu), introducing diagnostic uncertainty. Further investigations revealed severe cardiac involvement. Considering the recurring difficulty presented by the finding of a VUS in the GLA gene during FN assessments, along with the uncertainty regarding the need for treatment in nonclassical or late-onset FD phenotypes, especially in women, this case becomes a central focus for a thorough review of the literature. This review aims to propose a practical algorithm that integrates clinical, biochemical, and genetic markers for FN screening and diagnosis. Additionally, it explores treatment benefits in nonclassical or late-onset FD phenotypes, with a focus on female patients.
基金supported by grants from the National Natural Science Foundation of China(Grant Nos.82072602 and 82173222)the Science and Technology Commission of Shanghai Municipality(Grant Nos.20DZ2201900 and 18411953100)+1 种基金the Innovation Foundation of Translational Medicine of Shanghai Jiaotong University School of Medicine(Grant No.TM202001)the Collaborative Innovation Center for Clinical and Translational Science of the Chinese Ministry of Education&Shanghai(Grant No.CCTS-2022202)。
文摘Objective:Organoids are a powerful tool with broad application prospects in biomedicine.Notably,they provide alternatives to animal models for testing potential drugs before clinical trials.However,the number of passages for which organoids maintain cellular vitality ex vivo remains unclear.Methods:Herein,we constructed 55 gastric organoids from 35 individuals,serially passaged the organoids,and captured microscopic images for phenotypic evaluation.Senescence-associatedβ-galactosidase(SA-β-Gal),cell diameter in suspension,and gene expression reflecting cell cycle regulation were examined.The YOLOv3 object detection algorithm integrated with a convolutional block attention module(CBAM)was used to evaluate organoid vitality.Results:SA-β-Gal staining intensity;single-cell diameter;and expression of p15,p16,p21,CCNA2,CCNE2,and LMNB1 reflected the progression of aging in organoids during passaging.The CBAM-YOLOv3 algorithm precisely evaluated aging organoids on the basis of organoid average diameter,organoid number,and number×diameter,and the findings positively correlated with SA-β-Gal staining and single-cell diameter.Organoids derived from normal gastric mucosa had limited passaging ability(passages 1–5),before aging,whereas tumor organoids showed unlimited passaging potential for more than 45 passages(511 days)without showing clear senescence.Conclusions:Given the lack of indicators for evaluating organoid growth status,we established a reliable approach for integrated analysis of phenotypic parameters that uses an artificial intelligence algorithm to indicate organoid vitality.This method enables precise evaluation of organoid status in biomedical studies and monitoring of living biobanks.
基金supported by National Natural Science Foundation of China(Nos.22122803 and 21788102)the National Natural Science Foundation of Jiangsu Province(No.BK20220644).
文摘Aging-related diseases are gradually becoming a major problem with the rapid development of aged population in human society.Although many fluorescent probes have been employed to diagnosis senescence via imaging senescence-associatedβ-galactosidase(SA-β-Gal),which is proved to be closely associated with senescent cells,the similar catalytic effectiveness of enzymatic reaction of ovarian cancer-associatedβ-Gal(OA-β-Gal)will interfere with imaging accuracy.Herein,a near-infrared(NIR)hemicyanine based fluorescent probe HCyXA-βGal was designed for light-up imaging of live cells containingβ-Gal.With the organelle-targeting morpholinyl and positive charge moieties,HCyxA-βGal was successfully applicated to image the difference of enzymatic location in senescent cells and ovarian cancer cells.Furthermore,inspired by the fast response performance,fast and precise imaging of the two cell lines was realized via covering another dimension of fluorescence signal:time-dependent intensity.
基金supported by the National Natural Science Foundation of China(Grant No.30370913)the Guangdong Provincial Natural Science Foundation(Grant No.32264).
文摘To investigate the relationships among β-mannanase, β-mannosidase and a-galactosidase required for degrading galactomannan in cell wall during and following rice seed germination, the activities of the three enzymes and the effects of ABA and GA3 on them were surveyed. The activities of β-mannosidase and a-galactosidase presented in dry and pre-germinated rice seeds, and increased slowly during and following germination. However, the activity of β-mannanase was detected only after germination. GA3 could promote the activities of β-mannanase and a-galactosidase. ABA had little effect on the activities of β-mannosidase and α-galactosidase, but it could seriously inhibit the activity of β-mannanase.
文摘A retroviral vector pINC-lacZ containing an E coli β-galactosidase(β-gal)gene was constructed and introduced into the MESPU-13 cells by electroporation.Four G418-resistant colonies were obtained from 1×107,electroporated MESPU-13 cells. Histochemical staining for β-gal activity showed that lacZ gene was expressed in at least three of the four colonies.Southern analysis proved that one copy of foreign gene was integrated in the chromosome of one of the transformed lines(MC15).These results showed that the expression of lacZ gene driven by SiCMVIE promoter can be detected in the transformed MESPU-13 cells.
文摘β-galactosidases (EC 3.2.1.23) constitute a widespread family of glycosyl hydrolases in plants and are thought to be involved in metabolism of cell wall polysaccharides. A cDNA of the cotton (Gossypium hirsutum) β-galactosidase gene, designated GhGal1, has previously been identified and its transcripts are highly abundant at the elongation stage of the cotton fiber. To examine the temporal and spatial control of GhGal1 expression, a transcriptional fusion of the GhGal1 promoter region (1770 bp) with the β-glucuronidase (GUS) reporter gene was introduced into tobacco plants by the Agrobacterium infection method. The resulting transgenic plants showed higher GUS activity of fruit in the transgenic plants than that in the negative and positive controls. Histochemical localization of GUS activity demonstrated that the expression of the GUS gene could be found in the meristem zones of roots, cotyledons, vascular tissues, fruit and trichomes in transgenic tobacco plants. Additionally, se-quence analysis of the regulatory region also revealed several conserved motifs among which some were shared with previously reported fruit/seed-specific elements and the others were related with trichome expression. These results indicated the temporal and spatial expression characterization of the GhGal1 promoter in transgenic tobacco plants and provided an important insight into the roles of GhGal1 in cotton fiber development.
