A glycomic approach to hepatic tumors in N-acetylglucosaminyltransferase III (GnT-III) transgenic mice induced by diethylnitrosamine (DEN): identification of haptoglobin as a target molecule of GnT-III

A glycomic approach to the identification of target molecules in glycosyltransferase gene targeting mice is a promising strategy to understand the biological significance of glycosyltransferase genes in vivo . In order to understand the biological effects of N -acetylglucosaminyltransferase III (GnT-III) on tumor formation in the liver, diethylnitrosamine (DEN) induced tumor formation in the GnT-III transgenic mice was examined. Our findings show that the incidence of hepatic tumor could be dramatically suppressed. A glycomic approach using two-dimensional gel electrophoresis followed by lectin blot analysis and sequence analysis revealed that haptoglobin, a radical scavenger molecule in serum was heavily glycosylated in hepatic tumor-bearing GnT-III transgenic mice that had been treated with DEN. Immunoprecipitation followed by E 4 -PHA lectin blot analysis also confirmed that the bisecting GlcNAc, a product of GnT-III was added to haptoglobin molecules. Since the use of DEN is known to lead to the production of lipid peroxidation products which facilitate this reaction and haptoglobin is an acute phase reactant, acting as a radical scavenger against hemoglobin or iron stimulated lipid peroxidation, a relationship between the glycosylation of haptoglobin and the suppression of hepatoma development can not be ruled out. This paper is the first report that shows a relationship between the sugar chains of glycoproteins with radical scavenger activity and hepatocarcinogenesis.     

A Glycomic Approach to Hepatic Tumors in N -acetylglucosaminyltransferase III (GnT-III) Transgenic Mice Induced by Diethylnitrosamine (DEN): Identification of Haptoglobin as a Target Molecule of GnT-III

A glycomic approach to the identification of target molecules in glycosyltransferase gene targeting mice is a promising strategy to understand the biological significance of glycosyltransferase genes in vivo. In order to understand the biological effects of N -acetylglucosaminyltransferase III (GnT-III) on tumor formation in the liver, diethylnitrosamine (DEN) induced tumor formation in the GnT-III transgenic mice was examined. Our findings show that the incidence of hepatic tumor could be dramatically suppressed. A glycomic approach using two-dimensional gel electrophoresis followed by lectin blot analysis and sequence analysis revealed that haptoglobin, a radical scavenger molecule in serum was heavily glycosylated in hepatic tumor-bearing GnT-III transgenic mice that had been treated with DEN. Immunoprecipitation followed by E 4 -PHA lectin blot analysis also confirmed that the bisecting GlcNAc, a product of GnT-III was added to haptoglobin molecules. Since the use of DEN is known to lead to the production of lipid peroxidation products which facilitate this reaction and haptoglobin is an acute phase reactant, acting as a radical scavenger against hemoglobin or iron stimulated lipid peroxidation, a relationship between the glycosylation of haptoglobin and the suppression of hepatoma development can not be ruled out. This paper is the first report that shows a relationship between the sugar chains of glycoproteins with radical scavenger activity and hepatocarcinogenesis.     

Ectopic expression of alpha1,6 fucosyltransferase in mice causes steatosis in the liver and kidney accompanied by a modification of lysosomal acid lipase

The alpha1,6 fucosyltransferase (alpha1,6 FucT) catalyzes the transfer of a fucose from GDP-fucose to the innermost GlcNAc residue of N-linked glycans via an alpha1,6 linkage. alpha1,6 FucT was overexpressed in transgenic mice under the control of a combined cytomegalovirus and chicken beta-actin promoter. Histologically numerous small vacuoles, in which lipid droplets had accumulated, were observed in hepatocytes and proximal renal tubular cells. Electron microscopic studies showed that the lipid droplets were membrane-bound and apparently localized within the lysosomes. Cholesterol esters and triglycerides were significantly increased in liver and kidney of the transgenic mice. Liver lysosomal acid lipase (LAL) activity was significantly lower in the transgenic mice compared to the wild mice, whereas LAL protein level, which was detected immunochemically, was increased, indicating that the specific activity of LAL was much lower in the transgenic mice. In all of the transgenic and nontransgenic mice examined, the activity of liver LAL was negatively correlated with the level of alpha1,6 FucT activity. As evidenced by lectin and immunoblot analysis, LAL was found to be more fucosylated in the transgenic mice, suggesting that the aberrant fucosylation of LAL causes an accumulation of inactive LAL in the lysosomes. Such an accumulation of inactive LAL could be a likely cause for a steatosis in the lysosomes of the liver and kidney in the case of the alpha1,6 FucT transgenic mice.     

Remodeling of the major mouse xenoantigen, Galalpha1-3Galbeta1-4GlcNAc-R, by N-acetylglucosaminyltransferase-III

beta-D-Mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyses the attachment of an N-acetylglucosamine (GlcNAc) residue to mannose in the beta(1-4) configuration in N-glycans, and forms a bisecting GlcNAc. We have generated transgenic mice that contain the human GnT-III gene under the control of the mouse albumin enhancer/promoter [Lee et al., (2003)]. Overexpression of this gene in mice reduced the antigenicity of N-glycans to human natural antibodies, especially in the case of the alpha-Gal epitope, Galalpha1-3Galbeta1-4GlcNAc-R. Study of endothelial cells from the GnT-III transgenic mice revealed a significant reduction in antigenicity, and a dramatic decrease in both complement- and natural killer cell-mediated mouse cell lysis. Changes in the enzymatic activities of other glycosyltransferases, such as alpha1,3-galactosyltransferase, and alpha-6-D-mannoside beta-1,6 N-acetylglucosaminyltransferase V, did not point to any interaction between GnT-III and these enzymes in the transgenic mice, suggesting that this approach may be useful in clinical xenotransplantation.     

Transgenic mice aberrantly expressing human ornithine decarboxylase gene.

We have generated transgenic mice carrying human ornithine decarboxylase gene. Two different transgene constructs were used: (i) a 5'-truncated human ornithine decarboxylase gene and (ii) an intact human ornithine decarboxylase gene. Transgenic mice carrying the 5'-truncated gene did not express human ornithine decarboxylase-specific mRNA. Transgenic mice carrying the intact human ornithine decarboxylase gene expressed human-specific ornithine decarboxylase mRNA in all tissues studied. However, as indicated by actual enzyme assays, the expression pattern was highly unusual. In comparison with their wild-type littermates, the transgenic mice exhibited greatly elevated enzyme activity in almost every tissue studied. Ornithine decarboxylase activity was moderately elevated in parenchymal organs such as liver, kidney, and spleen. Tissues like heart, muscle, lung, thymus, testis, and brain displayed an enzyme activity that was 20 to 80 times higher than that in the respective tissues of nontransgenic animals. The offspring of the first transgenic male founder animal did not show any overt abnormalities, yet their reproductive performance was reduced. The second transgenic founder animal, showing similar aberrant expression of ornithine decarboxylase in all tissues studied, including an extremely high activity in testis, was found to be infertile. Histological examination of the tissues of the latter animal revealed marked changes in testicular morphology. The germinal epithelium was hypoplastic, and the spermatogenesis was virtually totally shut off. Similar examination of male members of the first transgenic mouse line revealed comparable, yet less severe, histological changes in testis.     

系统表达HB-EGF转基因小鼠的建立

目的建立系统表达肝素结合性表皮生长因子（HB-EGF）的转基因动物模型,利用转基因动物模型研究HB-EGF与组织纤维化的关系。方法RT-PCR法克隆小鼠HB-EGF基因,将其插入Chickenβ-actin启动子下游,构建Chickenβ-actin-HB-EGF表达载体,利用显微注射的技术把表达载体注射到受精卵的雄原核中,建立HB-EGF转基因小鼠。利用特异引物PCR的方法鉴定转基因的基因型,采用Western Blot方法鉴定HB-EGF基因在全身组织的表达。分别取HB-EGF转基因鼠与同窝阴性小鼠的肝、肾、肺及膀胱组织进行Massion染色。结果建立了系统表达HB-EGF转基因小鼠,Western Blot发现其HB-EGF在肝、肺、肾及膀胱的表达与同窝阴性对照小鼠相比明显增加。Massion染色结果表明转基因鼠肝、肺、肾及膀胱组织纤维化程度明显高于同窝阴性对照小鼠。结论成功建立了系统表达HB-EGF转基因小鼠,HB-EGF的过度表可显著加重组织纤维化程度。     

Break of T cell ignorance to a viral antigen in the liver induces hepatitis

To study peripheral tolerance of CD8 T cells to a classically MHC-restricted peptide Ag expressed in hepatocytes, ALB1 transgenic (tg) mice expressing the CTL epitope GP33 of the lymphocytic choriomeningitis virus glycoprotein under control of the mouse albumin promoter were generated. ALB1 mice exclusively expressed the GP33 transgene in the liver and, at a 100- to 1000-fold lower level, in the thymus. TCR-tg mice specific for the GP33 epitope were used to directly follow GP33-specific T cells in vivo. These experiments revealed that 1) thymic expression of the GP33 transgene led to incomplete central deletion of TCR-tg cells; and 2) peripheral TCR-tg cells in ALB1 mice ignored the GP33 transgene expressed in hepatocytes. Ignorance of adoptively transferred TCR-tg cells in ALB1 mice was broken by infection with lymphocytic choriomeningitis virus, leading to induction of hepatitis in ALB1, but not in control, mice. Taken together, we have established a novel model of virus-induced CD8 T cell-mediated autoimmune hepatitis in mice and demonstrate that naive CD8 T cells may ignore Ags expressed in the liver.     

Biosynthesis of high density lipoprotein by chicken liver: intracellular transport and proteolytic processing of nascent apolipoprotein A-1

To study the in vivo processing and secretion of Apolipoprotein A-I (Apo A-I), young chickens were administered individual L-[3H]amino acids intravenously and the time of intracellular transport of nascent Apo A-I from rough endoplasmic reticulum (RER) to the Golgi apparatus was measured. Within 3 to 9 min there was maximal incorporation of radioactivity into Apo A-I in both the RER and the Golgi cell fractions. By contrast, the majority of radioactive albumin was also present in the RER by 3 to 9 min, but did not reach peak amounts in the Golgi fraction until 9 to 25 min. Both radioactive Apo A-I and albumin appeared in the blood at about the same time (between 20 and 30 min). NH2-terminal amino acid sequence analysis of nascent intracellular Apo A-I showed that it contains a pro-hexapeptide extension identical to that of human Apo A-I. After 30 min of administration of radioactive amino acids radioactive Apo A-I was isolated by immunoprecipitation from the liver and serum. NH2-terminal sequence analysis of 20 amino acids indicated that chicken liver contained an equal mixture of nascent pro-Apo A-I and fully processed Apo A-I, whereas the serum only contained processed Apo A-I. Further studies showed that the RER only contained pro-Apo A-I, whereas a mixture of pro-Apo A-I and processed Apo A-I was found in the Golgi complex. These results indicate that, in chicken hepatocytes, there is a more rapid transport of Apo A-I than of albumin from the RER to the Golgi cell fractions, and that Apo A-I remains in the Golgi apparatus for a longer period of time before it is secreted into the blood. In addition these studies show that the in vivo proteolytic processing of chicken pro-Apo A-I to Apo A-I occurs in the Golgi cell fractions.     

Ectopically expressed PDX-1 in liver initiates endocrine and exocrine pancreas differentiation but causes dysmorphogenesis

To date, the potency of pancreatic and duodenal homeobox gene 1 (PDX-1) in inducing differentiation into insulin-producing cells has been demonstrated in some cells and tissues. In order to carry out efficient screening of somatic tissues and cells that can transdifferentiate into beta-cell-like cells in response to PDX-1, we generated CAG-CAT-PDX1 transgenic mice carrying a transgene cassette composed of the chicken beta-actin gene (CAG) promoter and a floxed stuffer DNA sequence (CAT) linked to PDX-1 cDNA. When the mice were crossed with Alb-Cre mice, which express the Cre recombinase driven by the rat albumin gene promoter, PDX-1 was expressed in more than 50% of hepatocytes and cholangiocytes. The PDX-1 (+) livers expressed a variety of endocrine hormone genes such as insulin, glucagon, somatostatin, and pancreatic polypeptide. In addition, they expressed exocrine genes such as elastase-1 and chymotrypsinogen 1B. However, the mice exhibited marked jaundice due to conjugated hyperbilirubinemia, and the liver tissue displayed abnormal lobe structures and multiple cystic lesions. Thus, the in vivo ectopic expression of PDX-1 in albumin-producing cells was able to initiate but not complete the differentiation of liver cells into pancreatic cells. The conditional PDX-1 transgenic mouse system developed in this study appeared to be useful for efficient screening of PDX-1 responsive somatic tissues and cells.     

CpG Oligodeoxynucleotides with Hepatitis B Surface Antigen (HBsAg) for Vaccination in HBsAg-Transgenic Mice

DNA motifs containing unmethylated CpG dinucleotides within the context of certain flanking sequences enhance both innate and antigen-specific immune responses, due in part to the enhanced production of Th1-type cytokines. Here we explored the ability of CpG-containing oligodeoxynucleotides combined with recombinant hepatitis B surface antigen (HBsAg) to induce Th1 responses in mice that are transgenic for this antigen and that represent a model for asymptomatic hepatitis B virus chronic carriers. This was compared to hepatitis B virus-specific DNA-mediated immunization, which we have previously shown to induce the clearance of the transgene expression product and the down-regulation of hepatitis B virus mRNA in this transgenic mouse lineage. In control nontransgenic C57BL/6 mice, three immunizations with HBsAg and CpG triggered the production of anti-HBs antibodies and of HBs-specific T cells that secrete gamma interferon but do not display any HBsAg-specific cytotoxic activity. In the HBsAg-transgenic mice, immunization with HBsAg and CpG oligodeoxynucleotides, but not with CpG alone, induced the clearance of HBsAg circulating in the sera, with a concomitant appearance of specific antibodies, and was able to regulate the hepatitis B virus mRNA constitutively expressed in the liver. Finally, adoptive transfer experiments with CD8(+) T cells primed in C57BL/6 mice with HBsAg and CpG oligodeoxynucleotide-based immunization show that these cells were able to partially control transgene expression in the liver and to clear the HBsAg from the sera of recipient transgenic mice without an antibody requirement. CpG oligodeoxynucleotides motifs combined with HBsAg could therefore represent a potential therapeutic approach with which to treat chronically infected patients.     

Generation of fad2 transgenic mice that produce omega-6 fatty acids

Fatty acid desaturase-2 (FAD2) introduces a double bond in position Δ12 in oleic acid (18︰1) to form linoleic acid (18︰2 n-6) in higher plants and microbes. A new transgenic expression cassette, containing CMV promoter/fad2 cDNA/SV40 polyA, was constructedto produce transgenic mice. Among 63 healthy offspring, 10 founders (15.9%) integrated the cotton fad2 transgene into their genomes, as demonstrated by PCR and Southern blotting analysis. All founder mice were fertile and heterozygous fad2 female and nontransgenic littermates were used for fatty acid analysis using gas chromatography. One fad2 transgenic line showed substantial differences in the fatty acid profiles and the level of linoleic acid was increased 19% (P<0.05) in transgenic muscles compared to their nontransgenic littermates. Moreover, it exhibited an 87% and a 9% increase (P<0.05) in arachidonic acid (20︰4 n-6) in muscles and liver, compared to their nontransgenic littermates. The results indicate that the plant fad2 gene can be functionally expressed in transgenic mice and may playan active role in conversion of oleic acid into linoleic acid.     

Increased milk yield in transgenic mice expressing insulin-like growth factor 1

Insulin-like growth factor 1 (IGF-1) mediates many of the actions of growth hormone. Overexpression of IGF-1 was reported to have endocrine and paracrine/autocrine effects on somatic growth in transgenic mice. To study the paracrine/autocrine effects of IGF-1 in mammary gland, transgenic mice were produced by pronuclear microinjection of a construct containing a bovine alpha-lactalbumin (alpha-LA) promoter linked to an ovine IGF-1 cNDA. This alpha-LA promoter has previously been shown to direct expression of a human factor VIII gene specifically to the mammary gland of transgenic mice. Three transgenic mouse lines were established as a result of microinjection of 398 embryos. Transgene expression was found in mammary gland at day 1 of lactation from these three lines. Progeny test were carried out by mating two transgenic males/one transgenic female to two nontransgenic females/one nontransgenic male. Mice from one line (line 1225) were all nonexpressors and the other (line 1372) failed to produce offspring. Milk yield was analyzed in the line 1137 that produced 10 mice, of which three were transgenic females and three nontransgenic females. All of the three transgenic females showed integration of the transgene and expressed transgene IGF-1 mRNA in the mammary gland. Milk yields from days 5, 10, and 15 of lactation were significant greater in transgenic expressors than in their nontransgenic littermates. Specifically, there is 17.9% increase in total milk yield from these three days for transgenics compared with nontransgenics. These results demonstrate that local overexpression of IGF-1 in transgenic mice is capable to stimulating milk yield during the first lactation.     

Antioxidant and oxidative status in tissues of manganese superoxide dismutase transgenic mice

Manganese superoxide dismutase (Mn-SOD) plays an important role in attenuating free radical-induced oxidative damage. The purpose of this research was to determine if increased expression of Mn-SOD gene alters intracellular redox status. Twelve week old male B6C3 mice, engineered to express human Mn-SOD in multiple organs, and their nontransgenic littermates were assessed for oxidative stress and antioxidant status in heart, brain, lung, skeletal muscle, liver, and kidney. Relative to their nontransgenic littermates, transgenic mice had significantly (p <.01) higher activity of Mn-SOD in heart, skeletal muscle, lung, and brain. Copper, zinc (Cu,Zn)-SOD activity was significantly higher in kidney, whereas catalase activity was lower in brain and liver. The activities of selenium (Se)-GSH peroxidase and non-Se-GSH peroxidase, and levels of vitamin E, ascorbic acid and GSH were not significantly different in any tissues measured between Mn-SOD transgenic mice and their nontransgenic controls. The levels of malondialdehyde were significantly lower in the muscle and heart of Mn-SOD mice, and conjugated dienes and protein carbonyls were not altered in any tissues measured. The results obtained showed that expression of human SOD gene did not systematical alter antioxidant systems or adversely affect the redox state of the transgenic mice. The results also suggest that expression of human SOD gene confers protection against peroxidative damage to membrane lipids.     

Hepatocellular kinetics and the expression of growth hormone (GH) in the livers and liver tumours of GH-transgenic mice

Although it is well known that transgenic mice that overexpress growth hormone (GH) frequently develop liver tumours, the precise nature of the relationship between the overexpression of GH and hepatocarcinogenesis is not clear. The current study was designed to investigate the relationship between the expression of the GH transgene and changes in hepatocyte morphology and kinetics, prior to and during hepatocarcinogenesis in GH-transgenic mice. In young mice (1-month-old) prior to tumour development, GH protein, as detected by immunohistochemistry, was observed in the cytoplasm of essentially all hepatocytes. In liver tissues of older animals, apoptotic cells and hypertrophic hepatocytes did not express immunoreactive GH, even though GH was expressed strongly in the smaller hepatocytes. A relatively high proportion of large dysplastic hepatocytes (>50 microm) were apoptotic (TUNEL positive), whereas smaller hepatocytes featured more prominently in the proliferative phase, as measured by BrdU incorporation. GH expression in tumour tissue, as detected by immunohistochemistry, was often variable and generally decreased with tumour development. Northern blot analysis showed that equivalent levels of GH mRNA were present in tumour tissue and adjacent liver. However, there was no clear trend when the levels of GH mRNA extracted from adenoma, and hepatocellular carcinoma, were compared. These observations help clarify some of the mechanisms by which GH promotes hepatocarcinogenesis in GH-transgenic mice.     

Inhibition of B cell death causes the development of an IgA nephropathy in (New Zealand white x C57BL/6)F(1)-bcl-2 transgenic mice

Little is known about the pathogenic mechanisms of IgA nephropathy, despite being the most prevalent form of glomerulonephritis in humans. We report in this study that in (New Zealand White (NZW) x C57BL/6)F(1) mice predisposed to autoimmune diseases, the expression of a human bcl-2 (hbcl-2) transgene in B cells promotes a CD4-dependent lupus-like syndrome characterized by IgG and IgA hypergammaglobulinemia, autoantibody production, and the development of a fatal glomerulonephritis. Histopathological analysis of glomerular lesions reveals that the glomerulonephritis observed in these animals resembles that of human IgA nephropathy. The overexpression of Bcl-2 in B cells selectively enhances systemic IgA immune responses to T-dependent Ags. Significantly, serum IgA purified from (NZW x C57BL/6)F(1)-hbcl-2 transgenic mice, but not from nontransgenic littermates, shows reduced levels of galactosylation and sialylation and an increased ability to deposit in the glomeruli, as observed in human patients with IgA nephropathy. Our results indicate that defects in the regulation of B lymphocyte survival associated with aberrant IgA glycosylation may be critically involved in the pathogenesis of IgA nephropathy, and that (NZW x C57BL/6)F(1)-hbcl-2 Tg mice provide a new experimental model for this form of glomerulonephritis.     

Increased Milk Yield in Transgenic Mice Expressing Insulin-like Growth Factor 1

Abstract

Insulin-like growth factor 1 (IGF-1) mediates many of the actions of growth hormone. Overexpression of IGF-1 was reported to have endocrine and paracrine/autocrine effects on somatic growth in transgenic mice. To study the paracrine/autocrine effects of IGF-1 in mammary gland, transgenic mice were produced by pronuclear microinjection of a construct containing a bovine α-lactalbumin (α-LA) promoter linked to an ovine IGF-1 cNDA. This α-LA promoter has previously been shown to direct expression of a human factor VIII gene specifically to the mammary gland of transgenic mice. Three transgenic mouse lines were established as a result of microinjection of 398 embryos. Transgene expression was found in mammary gland at day 1 of lactation from these three lines. Progeny test were carried out by mating two transgenic males/one transgenic female to two nontransgenic females/one nontransgenic male. Mice from one line (line 1225) were all nonexpressors and the other (line 1372) failed to produce offspring. Milk yield was analyzed in the line 1137 that produced 10 mice, of which three were transgenic females and three nontransgenic females. All of the three transgenic females showed integration of the transgene and expressed transgene IGF-1 mRNA in the mammary gland. Milk yields from days 5, 10, and 15 of lactation were significant greater in transgenic expressors than in their nontransgenic littermates. Specifically, there is 17.9% increase in total milk yield from these three days for transgenics compared with nontransgenics. These results demonstrate that local overexpression of IGF-1 in transgenic mice is capable to stimulating milk yield during the first lactation.     

Alteration in N -acetylglucosaminyltransferase activities and glycan structure in tissue and bile glycoproteins from extrahepatic bile duct carcinoma

The activities of three N -acetylglucosaminyltransferases (GnT)-III, IV and V, as well as the structural alterations of N-glycans on the glycoproteins in cancer tissues and bile specimens from 28 cases of extrahepatic bile duct carcinoma (EBDC) were compared with those from 18 cases of benign biliary duct diseases (BBDD). GnT activities were determined with fluorescence-labeled substrate using a HPLC method. It was found that GnT-III and GnT-V activities in EBDC were increased to 3.14 and 15.96 times respectively of the mean BBDD values, but GnT-IV remained unchanged. The activity of GnT-V was correlated with the grade of differentiation and TMN stage of EBDC. The up-regulation of GnT-III resulted in the increased bisecting-GlcNAc on the N-glycans of glycoproteins in cancer tissues and a 201 kDa bile glycoprotein when analyzed with HRP-labeled E(4)-PHA. The increased GnT-V activity led to the elevation of the beta1,6GlcNAc branch (or antennary number) on the N-glycans in cancer tissue glycoproteins and 201, 163, 122 kDa proteins in the bile as probed with HRP-labeled DSA. These findings suggest that the alteration in GnT activities may be involved in the malignant transformation and development of EBDC, resulting in the aberrant glycosylation of some tissue and bile proteins. The latter was expected to be used in the clinical diagnosis and prognosis evaluation in EBDC patients.