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Objective To investigate the establishment of a six-gene-edited pig-to-non-human primate kidney xenotransplantation model. Methods The kidney of humanized genetically-edited pig (GTKO/β4GalNT2KO/CMAHKO/hCD55/hCD46/hTBM) was transplanted into a cynomolgus monkey. The survival of the recipient and kidney condition after blood perfusion were observed. The parenchymal echo, blood flow changes, and size of the kidney were monitored on a regular basis. Routine blood test, kidney function test and electrolyte assessment were carried out. Dynamic changes of urine, feces and body mass were monitored. At the end of life, the transplant kidney, heart, liver, spleen, lung, and cecum were collected for pathological examination. Results The recipient died at postoperative 7 d. After blood flow was restored, the kidney was properly perfused, the organ was soft and the color was normal. At the end of the recipient's life, a slight amount of purulent secretion was attached to the ventral side of the kidney, with evident congestion and swelling, showing the appearance of "red kidney". Postoperatively, the echo of renal parenchyma was increased, blood flow was decreased, the cortex was gradually thickened, and a slight amount of effusion surrounded the kidney and abdominal cavity over time. In the recipient, the amount of peripheral red blood cells, hemoglobin, albumin, and platelets was progressively decreased, and serum creatinine level was increased to 308 μmol/L at postoperative 7 d, whereas the K+ concentration did not significantly change. Light yellow urine was discharged immediately after surgery, diet and drinking water were resumed within postoperative 3 h, and light yellow and normal-shape stool was discharged. The reddish urine was gradually restored to normal color within postoperative 1 d, which were consistent with the results of the routine urine test. A large amount of brown bloody stool was discharged twice in the morning of 2 d after surgery. Omeprazole was given for acid suppression, and the stool returned to normal at postoperative 4 d. The β2-microglobulin level was increased to 0.75 mg/L at postoperative 7 d. The body mass was increased by 1.7 kg. Autopsy pathological examination showed interstitial edema and bleeding of the transplant kidney, a large amount of infiltration of lymphocytes and macrophages, infiltration of lymphocytes in the arteriole wall and arterial cavity, accompanied by arteritis changes, lymphocyte infiltration in the cecal stroma and congestion in the spleen tissues. No significant abnormal changes were observed in other organs. Conclusions The humanized genetically-edited pig-to-non-human primate kidney xenotransplantation model is successfully established, and postoperative survival of the recipient is 1 week.
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Objective To evaluate the role and potential mechanism of interleukin (IL)-18/IL-18 binding protein (BP) in mediating the killing effect of natural killer (NK)-92MI cells upon endothelial cells from α-1, 3- galactosyltransferase gene-knockout (GTKO) porcine models. Methods NK-92MI cells were divided into the NK, NK+IL-18, NK+GTKO, IL-18+NK+GTKO and IL-18+IL-18BP+NK+GTKO groups. The messenger ribonucleic acid (mRNA) levels of inflammation-related genes in NK-92MI cells were detected by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The killing effect of NK-92MI cells on endothelial cells from GTKO porcine models was evaluated by lactate dehydrogenase (LDH) assay. The apoptosis of endothelial cells from GTKO porcine models was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. The expression levels of proteins with killing effect and apoptosis-related proteins were determined by Western blot. Results Compared with the NK, NK+IL-18 and NK+GTKO groups, the expression levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-8, IL-3, IL-6 and granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA were up-regulated in NK-92MI cells in the IL-18+NK+GTKO group, and the differences were statistically significant (all P < 0.05). Compared with the IL-18+NK+GTKO group, the expression levels of IFN-γ, TNF-α, IL-8, IL-3, IL-6 and GM-CSF mRNA were down-regulated in NK-92MI cells in the IL-18+IL-18BP+NK+GTKO group, and the differences were statistically significant (all P < 0.05). Compared with the NK+GTKO group, the expression levels of perforin, granzyme B and IFN-γ proteins in NK-92MI cells were up-regulated, the killing rate of NK-92MI cells against endothelial cells from GTKO porcine models was enhanced, the apoptosis rate of endothelial cells from GTKO porcine models was increased, and the ratios of B cell lymphoma-2 (Bcl-2)-associated X protein (Bax)/Bcl-2 and cleaved Caspase-3/Caspase-3 in endothelial cells from GTKO porcine models were elevated in the IL-18+NK+GTKO group, and the differences were statistically significant (all P < 0.05). Compared with the IL-18+NK+GTKO group, the expression levels of perforin, granzyme B and IFN-γ proteins were down-regulated, the killing rate of NK-92MI cells against endothelial cells from GTKO porcine models was decreased, the apoptosis rate of endothelial cells from GTKO porcine models was decreased, and the ratios of Bax/Bcl-2 and cleaved Caspase-3/Caspase-3 in endothelial cells from GTKO porcine models were declined in the IL-18+IL-18BP+NK+GTKO group, and the differences were statistically significant (all P < 0.05). Conclusions IL-18BP may block the expression of inflammation-related genes in NK-92MI cells induced by IL-18 and the killing effect of NK-92MI cells on endothelial cells from GTKO porcine models.
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ObjectiveThe effect of modified Shengjiangsan on immunoglobulin A （IgA） nephropathy was observed. The microRNA-148b （miRNA-148b）， interleukin 6 （IL-6）， core 1 beta 1，3-galactosyltransferase （C1GALT1）， molecular chaperone Cosmc （core1β3-Gal-T-specific molecular chaperone C1GALT1C1）， and galactose-deficient IgA1 （Gd-IGA1） in serum and kidney tissues of IgA nephropathy rats were detected to explore the underlying mechanism. The result is expected to lay a scientific basis for clinical application of modified Shengjiangsan in the treatment of IgA nephropathy. MethodA total of 42 SPF male SD rats were randomized into the normal group （8rats） and modeling group （34 rats） with the random number table method. After one week of adaptive feeding， rats for modeling were given bovine serum albumin （BSA， gavage）， lipopolysaccharide （LPS， injection into tail vein）， carbon tetrachloride （CCl4， subcutaneous injection）， and castor oil to induce IgA nephropathy. After modeling， two rats were randomly selected to test the modeling outcome. Then the model rats were classified into the model group， low-dose Chinese medicine group （modified Shengjiangsan，6.27 g·kg-1）， high-dose Chinese medicine group （modified Shengjiangsan，12.54 g·kg-1）， and benazepril group （10 mg·kg-1） with the random number table method， 8 in each group. The administration （gavage， once a day） lasted 4 weeks. The 24-h urinary total protein （24 h-UTP） was detected at the end of the 1st， 9th， and 13th week of the experiment. At the 14th week， after anesthesia， femoral artery blood was collected and centrifugated. The supernatant was collected to detect albumin （ALB）， aspartate aminotransferase （AST）， alanine aminotransferase （ALT）， serum creatinine （SCr）， and blood urea nitrogen （BUN）. The expression levels of IL-6 and Gd-IGA1 were determined by enzyme-linked immunosorbent assay （ELISA）. Based on hematoxylin-eosin （HE）/Masson/periodic Schiff-methenamine silver （PASM） staining， the pathological changes of renal tissues were observed. Ultrastructural changes of glomeruli were observed by transmission electron microscopy. The expression of miRNA-148b， IL-6， C1GALT1， and C1GALT1C1 was detected by immunohistochemistry. The mesangial area of the glomeruli was observed by immunofluorescence. Real-time polymerase chain reaction （Real-time PCR） was employed to determine the mRNA levels of mirNA-148b， IL-6， C1GALT1， and C1GALT1C1， and Western blot was used to detect the protein levels of IL-6， C1GALT1， and C1GALT1C1. ResultCompared with normal group， the model group showed increase in the content of 24 h-UTP， SCr， ALT， IL-6， and GD-IGA1 （P<0.05）， decrease in ALB content （P<0.05）. Moreover， rats in the model group demonstrated hyperplasia of glomerular mesangial cells， thickening of mesangial area， podocyte foot process effacement， and a large number of granular IgA immune complex in the mesangial area. In addition， the model group showed increase in the expression of IL-6 in mesangial area and podocytes， decrease in the expression of C1GALT1 and C1GALT1C1 in mesangial area and podocytes， enhanced expression of IL-6 mRNA and miRNA-148b （P<0.01）， weakened expression of C1GALT1 mRNA and C1GALT1C1 mRNA （P<0.01）， rise of IL-6 protein expression （P<0.01）， and reduction in the protein expression of C1GALT1 and C1GALT1C1 （P<0.01）. Compared with the model group， modified Shengjiangsan decreased the content of 24 h-UTP， SCr， ALT， IL-6， and Gd-IGA1 （P<0.05） and increased the content of ALB （P<0.05， P<0.01）. Moreover， with the treatment of this Chinese medicine， the pathological damage was significantly alleviated and the deposition of IgA immune complex in basement membrane was reduced. The expression of IL-6 in the mesangial area and podocytes of rats was decreased， and the expression of C1GALT1 and C1GALT1C1 in the mesangial area and podocytes of rats was increased. Moreover， the expression of IL-6 mRNA and miRNA-148b was decreased （P<0.01）， and the expression of C1GALT1 mRNA and C1GALT1C1 mRNA was increased （P<0.01）. The protein expression of IL-6 was decreased （P<0.05， P<0.01）， and the protein expression of C1GALT1 and C1GALT1C1 was enhanced （P<0.05， P<0.01）. The Chinese medicine group showed obvious dose-effect trend. ConclusionModified Shengjiangsan may reduce the expression of miRNA-148b and IL-6 in serum and kidney tissue of IgA nephropathy rats， restore the expression of C1GALT1 and C1GALT1C1， and decrease the generation of Gd-IGA1， so as to reduce renal pathological damage and proteinuria， protect the kidney protection， and finally delay the disease progression. Moreover， the effect is enhanced with the rise of dose.
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Objective: Myricetin 3-O-galactoside is an active compound with pharmaceutical potential. The insufficient supply of this compound becomes a bottleneck in the druggability study of myricetin 3-O-galactoside. Thus, it is necessary to develop a biosynthetic process for myricetin 3-O-galactoside through metabolic engineering. Methods: Two genes OcSUS1 and OcUGE1 encoding sucrose synthase and UDP-glucose 4-epimerase were introduced into BL21(DE3) to reconstruct a UDP-D-galactose (UDP-Gal) biosynthetic pathway in Escherichia coli. The resultant chassis strain was able to produce UDP-Gal. Subsequently, a flavonol 3-O-galactosyltransferase DkFGT gene was transformed into the chassis strain producing UDP-Gal. An artificial pathway for myricetin 3-O-galactoside biosynthesis was thus constructed in E. coli. Results: The obtained engineered strain was demonstrated to be capable of producing myricetin 3-O-galactoside, reaching 29.7 mg/L. Conclusion: Biosynthesis of myricetin 3-O-galactoside through engineered E. coli could be achieved. This result lays the foundation for the large-scale preparation of myricetin 3-O-galactoside.
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Objective To investigate the mechanism underlying the activation of tissue factor (TF) that leads to coagulation dysfunction in the recipients after liver xenotransplantation. Methods Auxiliary heterotopic liver xenotransplantation was performed in 3 minipigs with α-1,3-galactosyltransferase gene-knockout (GTKO) as the donors and Tibetan macaque (Macaca thibetana) as the recipients. Postoperative coagulation function changes in the recipients were observed. Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining were adopted to quantitatively measure the expression levels of monkey and minipig TF messenger RNA (mRNA) and protein in the liver tissues of the primary and transplant livers at different time points before and after transplantation. The recalcification time of peripheral blood mononuclear cell (PBMC) was recorded in the normal control monkeys and the recipient monkeys before and 2 h after liver transplantation to evaluate the coagulation status in the recipients. Results All three recipients presented with different degrees of coagulation dysfunction after surgery, manifested as a decrease in fibrinogen level and a reduction in platelet count. The monkey TF protein was positively expressed in the primary livers after surgery, whereas negatively expressed in transplant livers before and after liver transplantation. The minipig TF protein was negatively expressed in both primary livers and transplant livers. At postoperative 2 h, monkey TF mRNA was up-regulated by (2.10±0.24) times in the primary liver compared with the preoperative level, whereas the minipig TF mRNA was up-regulated by (1.42±0.15) times compared with preoperative level. There was statistical significance between the primary livers and transplant livers (P=0.014). Compared with PBMC in the normal control monkeys and recipient monkeys before liver transplantation, the recalcification time of the PBMC in the recipient monkeys was significantly shortened at postoperative 2 h (both P<0.001). Conclusions At the presence of coagulation dysfunction after liver xenotransplantation, the level of TF activation in the primary livers is significantly higher than that in the transplant livers. The TF activation in the primary livers is the main cause of coagulation dysfunction after liver xenotransplantation.
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Objective:To investigate the effects and mechanism of Ginseng polysaccharides (GPS) on glycosaminoglycan synthesis in rat chondrocytes in vitro.Methods:Chondrocytes isolated from knee joint cartilage of rats were subcultured.After monolayers were established,chondrocytes induced by 20 ng·ml-1 IL-1β were treated with GPS respectively at the concentration of 2,10 and 50 ng ·ml-1for 48 h.The effects of GPS on chondrocytes were analyzed using MTT assay for chondrocyte proliferation and 1,9-dimethylmethylene blue assay for GAG content.mRNA content in xylosyltransferase-Ⅰ(XylT-Ⅰ),galactosyltransferase-Ⅰ(GalT-Ⅰ),galactosyltransferase Ⅱ(GalT-II) and galactose-β-1,3-glucuronosyltransferase Ⅰ(GlcAT-Ⅰ) and that in matrix-degrading enzymes matrix metalloproteinases-3 (MMP-3) and aggrecanase-1and aggrecanase-2 was also detected.Results:GPS had no toxic or proliferating effect on chondrocytes at all observed concentrations.GPS at the concentrations of 10 ng·ml-1 and 50 ng·ml-1 up-regulated GAG synthesis (P<0.01).10 ng·ml-1 GPS enhanced the mRNA expression of XylT-Ⅰ (P<0.01),and 50 ng·ml-1 GPS enhanced the mRNA expression of XylT-Ⅰ,GalT-Ⅰ and GalT-II (P<0.05,P<0.01).GPS did not inhibit the mRNA expression of matrix-degrading enzymes MMP-3 and aggrecanase-1and aggrecanase-2 enhanced by IL-1β.Conclusion:GPS can improve GAG synthesis in IL-1β stimulated chondrocytes in vitro,which is due to the expression promotion of GAG synthesis enzymes without the expression inhibition of matrix-degrading enzymes.
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Objective To investigate the optimal condition for the detection of anti-non-galactose (Gal) xenoantigen and antibody in human serum.Mehtods Peripheral blood mononuclear cell (PBMC) obtained from Wuzhishan miniature pig models with α-1,3-galactosyltransferase gene knockout (GTKO) were used as target cells,mixed and incubated with healthy human serum of different concentrations (4.8％,16.7％ and 100％) for 0.5,1.0,2.0,3.0 and 6.0 h,respectively.The abilities of PBMC to bind with IgM and IgG were detected by flow cytometry.Results At the serum concentration of 16.7％,the ability ofnon-Gal IgM to bind with PBMC was significantly enhanced from 0.5 h to 3.0 h incubation (P＜0.01),whereas no statistical significance was noted in terms of IgG (P＞0.05).Increasing serum concentration could also enhance the ability of non-Gal IgM to bind with PBMC.At the serum concentration of 100％ and incubation for 3 h,the ability of IgM to bind with PBMC was the highest among all groups (P＜0.01).At the serum concentration of 100％ and incubation for 6 h,the ability of IgG to bind with PBMC was significantly enhanced (P＜0.05).Prolonging incubation time and increasing serum concentration did not affect the activity of PBMC.Conclusions The optimal condition for detection of anti-non-Gal xenoantigen and antibody is determined.A quantity of 1×105 PBMC from pig should be incubated with 100％ human serum for 3 h for detection of IgM level,or incubated with 100％ human serum for 6 h for measurement of IgG level.This optimized condition contributes to screening the donor pigs which lowly express non-Gal antigen.
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To summarize the breeding and identification of Wuzhishan miniature pig models with α-l,3-galactosyltransferase (GGTA1) gene-knockout (GTKO).Methods The breeding and reproduction perform of GTKO Wuzhishan miniature pigs were assessed and the quantity of piglets was counted.The GTKO Wuzhishan miniature pig models with GGTA1gene knockout were validated by polymerase chain reaction(PCR).The αGal phenotype of peripheral blood mononuclear cells (PBMC) in human,wild-type Wuzhishan miniature pigs and GTKO Wuzhishan miniature pigs was detected by fluorescent microscope and flow cytometry.Routine blood test parameters were statistically compared between the GTKO and wild-type Wuzhishan miniature pigs.Results The inheritance of GGTA1 genotype complied with Mendel's law.Flow cytometry detected no fluorescent expression of PBMC in GGTA1-/-pig models,which were consistent with the genotype identification results.The mean piglets of the primiparous GTKO Wuzhishan miniature pigs were (6.8±1.8) and (8.3±2.2) for the multiparous Wuzhishan miniature pigs.No statistical significance was noted in routine blood test parameters between the GTKO and wild-type Wuzhishan miniature pigs (all P＞0.05).Conclusions Stable inheritance and normal reproductive capacity are observed in two generations of Wuzhishan miniature pigs continuously.GTKO Wuzhishan miniature pig is a reliable donor for heterogeneous organ transplantation.
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Recent developments in genome editing technology using meganucleases demonstrate an efficient method of producing gene edited pigs. In this study, we examined the effectiveness of the transcription activator-like effector nuclease (TALEN) system in generating specific mutations on the pig genome. Specific TALEN was designed to induce a double-strand break on exon 9 of the porcine α1,3-galactosyltransferase (GGTA1) gene as it is the main cause of hyperacute rejection after xenotransplantation. Human decay-accelerating factor (hDAF) gene, which can produce a complement inhibitor to protect cells from complement attack after xenotransplantation, was also integrated into the genome simultaneously. Plasmids coding for the TALEN pair and hDAF gene were transfected into porcine cells by electroporation to disrupt the porcine GGTA1 gene and express hDAF. The transfected cells were then sorted using a biotin-labeled IB4 lectin attached to magnetic beads to obtain GGTA1 deficient cells. As a result, we established GGTA1 knockout (KO) cell lines with biallelic modification (35.0%) and GGTA1 KO cell lines expressing hDAF (13.0%). When these cells were used for somatic cell nuclear transfer, we successfully obtained live GGTA1 KO pigs expressing hDAF. Our results demonstrate that TALEN-mediated genome editing is efficient and can be successfully used to generate gene edited pigs.
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Animales , Humanos , Antígenos CD55/genética , Línea Celular , Roturas del ADN de Doble Cadena , Exones/genética , Galactosiltransferasas/genética , Edición Génica/veterinaria , Técnicas de Inactivación de Genes , Técnicas de Transferencia Nuclear , Porcinos , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genéticaRESUMEN
Objective To investigate the altered glycotransferases and glycan profile in HCV-infected cells, the expression levels of 35 kinds of sialyltransferases and galactosyltransferases were evaluated in hepatitis C virus (HCV) infected Huh7.5.1 cell cultured model.Methods Western blot was used to measure the expressions of HCV-nonstructural protein NS3 and and quantitative real-time PCR (qRT-PCR) was performed to determine HCV-RNA levels and mRNA levels of 35 kinds of sialyltransferases and galactosyltransferases.Lectin microarray was used to compare the glycan profiles of sialyltransferases-and galactosyltransferases-associated glycan-profile in Huh7.5.1 cells and HCV infected Huh7.5.1 cells.Results Among the 35 kinds of sialyltransferases and galactosyltransferases, the mRNA levels of four sialyltransferases (including ST3Gal Ⅲ, ST6GalNAC Ⅲ, ST8Sia Ⅲ and ST8SiaⅣ) and five galactosyltransferases (including B3GALT1, B3GALT 2, B3GALT3, B3GALT4 and B4GALNT2) were found to be 11-45 fold higher in HCV-infected cells compared with naive Huh7.5.1 cells.The up-regulated level of B3GALT 1 was the most evident (45-fold change), followed by ST8Sia Ⅳ and ST8Sia Ⅲ, with 39-and 37-fold respectively.Consistently, lectin microarray showed that glycans recognized by EBL (binding terminal sialic acid, Neu5Acα6Gal, 1.97-fold change), ECA letin (binding terminal galactose, Galβ1,4GalNAc,4.3-fold change), and ACA(binding terminal galactose, Galβ1-3GalNAc, 3-fold change) were elevated in glycoprotein products of sialyltransferase and galactosyltransferase respectively.Conclusion HCV infection causes the increased expression levels of mutiple sialyltransferases and galactosyltransferases in Huh7.5.1 cell line and hence the upregulation of glycan profiles of the glycoproteins.These results provide potential therapeutic targets and diagnostic markers for HCV infection and insights on the molecular mechanism of HCV infection.
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Objective To analyze whether β1,4-galactosyltransferase-Ⅰ(β1,4-GaiT-Ⅰ)expression correlates with the expression of tumor necrosis factor(TNF)-α in osteoarthritis(OA).Methods Synovial tissue samples from eight OA patients and eight healthy people were obtained as the experimental group and controls respectively.The mRNA levels of β1,4-GalT-Ⅰ and TNF-α were measured by reverse transcriptionpolymerase chain reaction(RT-PCR)and real-time PCR.Enzyme linked immunosorbent assay(ELISA)was used to test the expression of TNF-α in the protein level.Cellular colocalization of β1,4-GalT-Ⅰ and TNF-α was analyzed by double immunofluorescence.ANOVA and t-test was used for statistical analysis.Results ①Compared with the control group[β1,4-GalT-Ⅰ(0.48±0.09),TNF-α(0.46±0.07)],the expression of β1,4-GalT-Ⅰ(0.94±0.16)and TNF-α(1.19±0.19)were significantly increased in OA synovial tissue(t=3.47,t=4.06,P＜0.01)and there was colocalization between β1,4-GalT-Ⅰ and TNF-α;② Lipopolysaccharide (LPS)could induce fibroblast-like synoviocytes(FLSs)β1,4-GalT-Ⅰ[11.2±0.9 vs 2.9±0.5(dose effect),22.3±2.3 vs 4.4±0.9(time effect),F=83.03,F=157.58,P＜0.05]overexpression;③ LPS could induce FLSs TNF-α[(1256±96)vs(101±7)pg/ml,F=431.96,P＜0.01]overexpression;④ Not only endogenous TNF-α,but exogenous TNF-α could induce FLSs β1,4-GalT-Ⅰ[23.2±1.9 vs 8.4±1.3(dose effect),23.9±1.8 vs 11.5±1.3(time effect),F=124,F=93.6,P＜0.05]overexpression.Conclusion It is possible that FLSs mayuse TNF-αto control β1,4-GalT-Ⅰ functions during inflammation in OA.
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Xenotransplantation is a feasibility way of solving the shortage of human organs for transplantation. Although it is urgently needed to satisfy the demand of people and sustain the function of human organs, there are multiple hurdles existed to clinical application, such as the immune rejection between human body and the xenografts, the infection of pathogens and a series of ethic, morality and social issues. A historical retrospect of xenotransplantation was given, and then probe into the strategies according to the main problems and the actualities. Finally, the prospect in the field of xenotransplantation was showed.
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Activities of cytidine 5'-diphosphate-choline glycerol choline phosphotransferase and uridine 5'-diphosphate galactose-ceramide galactosyltransferase were determined in isolated myelin in different brain regions of control, and rats with restricted food intake. Kinetic experiments indicated an increase in Km value of phosphocholinetransferase in brain stem of undernourished rats, without significant change in the specific activity of this enzyme. Stimulation of this myelin bound enzyme activity was also evident in the animals when myelin was treated with the detergent: Tween CF. 54. Though specific activities of galactosyl transferase in myelin of undernourished rats were significantly diminished, the Km of this enzyme was unaltered. These studies point to an adverse effect of early nutritional stress on the activities of enzymes bound to myelin membrane which has hitherto been considered metabolically inert.
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Objective:To express and purificate catalytic domain of murine?-1,3-galactosyltransferase and provide the feasible method in the tumor cell surface production ?-gal epitopes..Methods:This research established expression system in pET-15b to express catalytic domain of murine ?-1,3-galactosyltransferase,then identified its activity by HPLC with anion exchange column.Results:(1)Constructed successfully recombinant ?-1,3-galactosyltransferase catalytic domain with His-tag.(2)?-1,3-galactosyltransferase with His-tag in a soluble form was expressed and purificated effeciently.(3)Its activity by HPLC with anion exchange column showed.Conclusion:This research shows ?-1,3-galactosyltransferase in a soluble form has been expressed successfully.
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The full length cDNA of ?1,3 galactosyltransferase gene was amplified by RT PCR from the peritoneal macrophages of BALB/c mice, which was subcloned into an eukaryotic expression vector pCDNA3 1/V5 HisA, and it was confirmed by DNA sequencing. The gastric cancer cell GC9811 was transfected with pCDNA3 1/V5 HisA?1,3GT by Lipofectamine TM 2000. The positive clone was screened by G418 for two months and confirmed by RT PCR. The gastric cells expressing Gal?(1,3)Gal epitope were detected by immuohistochemisty, and they were killed by naive antidoby specific to Gal?(1,3)Gal epitope. The results showed that the pCDNA3.1/V5 HisA?1,3GT was successfully constructed, and the transfected gastric cancer cells could highly express Gal?(1,3)Gal, which promoted lysis of the cancer cells by normal human serum.
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Buffalo milk galactosyltransferase is a single poly-peptide of molecular weight 55,000 to 56,000. The enzyme is specific for glucose as an acceptor substrate in the presence of ∝-lactalbumin, L-Arabinose. L-xylose, D-ribose and D-fructose did not serve as acceptor substrates even at concentration as high as 0.13 Μ, while N-acetylglucosamine and ovalbumin served as good acceptors of galactosyl moiety in the absence of ∝ -lactalbumin. UDP-galacturonic acid did not serve as a donor substrate; on the contrary, it inhibited the reaction. Lactose synthetase reaction was inhibited by D-ribose, L-arabinose and L-xylose, whereas D-fructose did not show any inhibition. Buffalo milk ∝ -lactalbumin enhanced the synthesis of lactose but inhibited the synthesis of N-acetyllactosamine. Cations like Ca2+ , Mg2+ , Cu2+ , Ba2+ and Co2+ could not replace Mn2+ in the N-acetyllactosamine synthetase reaction. Except Co2+ , these cations had no effect on this reaction. Co2+ was found to be a competitive inhibitor of Mn2+ . The observed inhibition of the reaction by·EDTA also confirmed the absolute requirement of Mn2+ for the reaction. Lactose synthetase reaction had an optimum pH of 8.5, whereas N-acetyllactosamine synthetase reaction was maximal at pH 8.0.
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Objective In order to study the effect of ? 1,4 GalT Ⅰ on proliferation of Schwann cells, The changes in cell proliferation were checked after Schwann cells transfected with sense or antisense ? 1,4 GalT Ⅰ plasmids. Methods Methods of counting numbers of proliferating cell and measuring [ 3H] thymidine incorporation by liquid scintillation were used to study the proliferation of purified Schwann cells which transfected with ? 1,4 GalT Ⅰ plasmids. Cell cycles of those transfected cells were determined by fluorescence activated cell sorting. Results After Schwann cells transfected with sense ? 1,4 GalT Ⅰ plasmids, the proliferation of those cells was restrained, and the number at G 1/G 0 phase of those cells increased while the number at S phase decreased. Schwann cells transfected with antisense ? 1,4 GalT Ⅰ plasmid showed opposite changes.Conclusion ? 1,4 GalT Ⅰ may play an important role in regulating the proliferation of Schwann cells in peripheral nerve.\;[