Attempting to Convert Primed Porcine Embryonic Stem Cells into a Naive State Through the Overexpression of Reprogramming Factors.

Establishing pig embryonic stem cells (pESCs) remains a challenge due to differences in the genetic backgrounds of mouse, human, and pig. Therefore, pig-specific pluripotency markers and cellular signaling must be identified. In this study, doxycycline (DOX)-inducible vectors carrying Oct4, sex-determining region Y-box 2 (Sox2), Nanog, Kruppel-like family 4 (Klf4), or Myc, which are known reprogramming factors, were transduced into pESCs. And pluripotency genes were analyzed in one or two reprogramming factor-expressed pESCs. When cultured without DOX, pESCs were stably maintained in basic fibroblast growth factor-supplemented media. However, when treated with DOX, the cells lost their alkaline phosphatase (AP) activity and differentiated within 2 weeks. Subsequently, we investigated the expression of genes related to pluripotency in DOX-treated pESCs using quantitative reverse transcription-polymerase chain reaction (PCR). Expression levels of Oct4, E-cadherin, and Fut4 were significantly increased by Oct4 overexpression, and Oct4 and Fut4 were upregulated in the Sox2-infected group. When a combination of two reprogramming factors, including Oct4 or Sox2, was introduced, weak AP activity remained. In addition, several of the two reprogramming factor transduction groups could be maintained after subculturing with transgene activation. Although long-term culture failed, pESCs transduced with Oct4 and Nanog, Oct4 and Klf4, or Sox2 and Nanog combinations could be subcultured even under transgene activation conditions. Analysis of the cause of long-term culture failure by quantitative PCR confirmed that the expression of intermediate reprogramming markers was not maintained. Given these results, additional methods are needed to support the completion of each reprogramming phase to succeed in the conversion of the pluripotent state of pESCs. This study improves our understanding of pluripotent networks and can be used to aid in the establishment of bona fide pig pluripotent stem cells.

New observations on the utility of CA19-9 as a biomarker in Lewis negative patients with pancreatic cancer.

BACKGROUND: Carbohydrate antigen 19-9 (CA19-9) is the best-validated biomarker for pancreatic cancer. The National Comprehensive Cancer Network (NCCN) guideline asserts that "CA19-9 will be undetectable in Lewis antigen-negative individuals". However, reports of CA19-9 secretion and its significance in Lewis (-) patients with pancreatic cancer have been inconsistent. This study was to examine serum CA19-9 levels in patients with pancreatic cancer according to Lewis status. METHODS: Patients with pancreatic cancer (1482 cases) were retrieved from a prospectively maintained database. Patients with benign pancreatic disease (210 cases) and normal subjects (315 cases) were used as controls. Lewis genotypes were examined by fucosyltransferase 3 (FUT3) sequencing. RESULTS: In patients with pancreatic cancer, 8.4% of subjects were Lewis (-), but only 41.9% of Lewis (-) subjects had CA19-9 values ≤ 2 U/mL. CA19-9 was even elevated (>37 U/mL) in 27.4% of Lewis (-) patients. The area under the receiver operating characteristic (ROC) curve for CA19-9 as a diagnostic biomarker was 0.842 in Lewis (-) patients with pancreatic cancer, which is closing to that of CA19-9 applied in all of patients with pancreatic cancer (0.898). Lewis (-) status was an independent prognostic factor for shorter survival in a multivariable analysis (hazard ratio (HR), 1.30, 95% confidence interval (CI), 1.03-1.64; P = 0.028). CONCLUSIONS: Not all Lewis (-) patients with pancreatic cancer are non-secretors of CA19-9. Contrary to general understanding, CA19-9 can retain its utility as a biomarker in these patients in spite of Lewis (-) genotype.

Lack of the α1,3-Fucosyltransferase Gene (Osfuct) Affects Anther Development and Pollen Viability in Rice.

N-linked glycosylation is one of the key post-translational modifications. α1,3-Fucosyltransferase (OsFucT) is responsible for transferring α1,3-linked fucose residues to the glycoprotein N-glycan in plants. We characterized an Osfuct mutant that displayed pleiotropic developmental defects, such as impaired anther and pollen development, diminished growth, shorter plant height, fewer tillers, and shorter panicle length and internodes under field conditions. In addition, the anthers were curved, the pollen grains were shriveled, and pollen viability and pollen number per anther decreased dramatically in the mutant. Matrix-assisted laser desorption/ionization time-of-flight analyses of the N-glycans revealed that α1,3-fucose was lacking in the N-glycan structure of the mutant. Mutant complementation revealed that the phenotype was caused by loss of Osfuct function. Transcriptome profiling also showed that several genes essential for plant developmental processes were significantly altered in the mutant, including protein kinases, transcription factors, genes involved in metabolism, genes related to protein synthesis, and hypothetical proteins. Moreover, the mutant exhibited sensitivity to an increased concentration of salt. This study facilitates a further understanding of the function of genes mediating N-glycan modification and anther and pollen development in rice.

Establishment of a tobacco BY2 cell line devoid of plant-specific xylose and fucose as a platform for the production of biotherapeutic proteins.

Plant-produced glycoproteins contain N-linked glycans with plant-specific residues of ß(1,2)-xylose and core α(1,3)-fucose, which do not exist in mammalian-derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant-expressed proteins. We knocked out the ß(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. In total, we knocked out 14 loci. The knocked-out lines were stable, viable and exhibited a typical BY2 growing rate. Glycan analysis of the endogenous proteins of these lines exhibited N-linked glycans lacking ß(1,2)-xylose and/or α(1,3)-fucose. The knocked-out lines were further transformed successfully with recombinant DNaseI. The expression level and the activity of the recombinant protein were similar to that of the protein produced in the wild-type BY2 cells. The recombinant DNaseI was shown to be totally free from any xylose and/or fucose residues. The glyco-engineered BY2 lines provide a valuable platform for producing potent biopharmaceutical products. Furthermore, these results demonstrate the power of the CRISPR/Cas9 technology for multiplex gene editing in BY2 cells.

Ginsenoside Rg3 inhibits epithelial-mesenchymal transition (EMT) and invasion of lung cancer by down-regulating FUT4.

The epithelial-mesenchymal transition (EMT) is an important factor in lung cancer metastasis, and targeting EMT is a potential therapeutic strategy. Fucosyltransferase IV (FUT4) and its synthetic cancer sugar antigen Lewis Y (LeY) was abnormally elevated in many cancers. In this study, a traditional Chinese medicine ginsenoside Rg3 was used to investigate whether its inhibition to EMT and invasion of lung cancer is by the glycobiology mechanism. We found that Rg3 treatment (25, 50, 100 µg/ml) inhibited cell migration and invasion by wound-healing and transwell assays. Rg3 could significantly alter EMT marker proteins with increased E-cadherin, but decreased Snail, N-cadherin and Vimentin expression. Rg3 also down-regulated FUT4 gene and protein expression in lung cancer cells by qPCR, Western blot and immunofluorescence. After FUT4 down-regulated with shFUT4, EMT was obviously inhibited. Furthermore, the activation of EGFR through decreased LeY biosynthesis was inhibited, which blocked the downstream MAPK and NF-κB signal pathways. In addition, Rg3 reduced tumor volume and weight in xenograft mouse model, and significantly decreased tumor metastasis nodules in lung tissues by tail vein injection. In conclusion, Rg3 inhibits EMT and invasion of lung cancer by down-regulating FUT4 mediated EGFR inactivation and blocking MAPK and NF-κB signal pathways. Rg3 may be a potentially effective agent for the treatment of lung cancer.

Ginsenoside Rg3 suppresses FUT4 expression through inhibiting NF-κB/p65 signaling pathway to promote melanoma cell death.

Abnormal glycosylation is catalyzed by the specific glycosyltransferases and correlates with tumor cell apoptosis. Increased fucosyltransferase IV (FUT4) is seen in many types of cancer, and manipulating FUT4 expression through specific signaling pathway inhibits cell growth and induces apoptosis. NF-κB is known playing a vital role to control cell growth and apoptosis. Ginsenoside Rg3 is an herbal medicine with strong antitumor activity through inhibiting tumor growth and promoting tumor cell death. However, whether Rg3-induced inhibition on tumor development involves reduced NF-κB signaling and FUT4 expression remains unknown. In the present study, we found that Rg3 suppressed FUT4 expression by abrogating the binding of NF-κB to FUT4 promoter through inhibiting the expression of signaling molecules of NF-κB pathway, reducing NF-κB DNA binding activity and NF-κB transcription activity. NF-κB inhibitor (Bay 11-7082) or knocking down p65 expression by p65 siRNA also led to a significant decreased FUT4 expression. In addition, Rg3 induced apoptosis by activating both extrinsic and intrinsic apoptotic pathways. Moreover, in a xenograft mouse model, Rg3 downregulated FUT4 and NF-κB/p65 expression and suppressed melanoma cell growth and induced apoptosis without any noticeable toxicity. In conclusion, Rg3 induces tumor cell apoptosis correlated with its inhibitory effect on NF-κB signaling pathway-mediated FUT4 expression. Results suggest Rg3 might be a novel therapy agent for melanoma treatment.

Ginsenoside Rg3-induced EGFR/MAPK pathway deactivation inhibits melanoma cell proliferation by decreasing FUT4/LeY expression.

Malignant melanoma is a destructive and lethal form of skin cancer with poor prognosis. An effective treatment for melanoma is greatly needed. Ginsenoside Rg3 is a herbal medicine with high antitumor activity. It is reported that abnormal glycosylation is correlated with the tumor cell growth. However, the antitumor effect of Rg3 on melanoma and its mechanism on regulating glycosylation are unknown. We found that Rg3 did not only inhibit A375 melanoma cell proliferation in a dose-dependent manner, but also decreased the expression of fucosyltransferase IV (FUT4) and its synthetic product Lewis Y (LeY), a tumor-associated carbohydrate antigen (TACA). Knocking down FUT4 expression by siRNA dramatically reduced FUT4/LeY level and inhibited cell proliferation through preventing the activation of EGFR/MAPK pathway. Consistently, the inhibitory effect of the Rg3 and FUT4 knockdown on melanoma growth was also seen in a xenograft melanoma mouse model. In conclusion, Rg3 effectively inhibited melanoma cell growth by downregulating FUT4 both in vitro and in vivo. Targeting FUT4/LeY mediated fucosylation by Rg3 inhibited the activation of EGFR/MAPK pathway and prevented melanoma growth. Results from this study suggest Rg3 is a potential novel therapy agent for melanoma treatment.

Increased thermosensitivity of mouse colorectal carcinoma cells transfected with human FUT1 gene.

The thermal responses of mouse colorectal carcinoma cells were investigated in the wild type cells and the transfected cells with human FUT1 gene which encodes alpha 1,2fucosyltransferase. The heat sensitivity was observed to increase in the FUT1 gene transfected cells and the effect of hyperthermia at 44 degrees C on these cells was demonstrated to be significant (P<0.001) to the wild type cells even though no remarkable difference in the expression of the heat shock protein, Hsp70 was found in these cells. Thus the expression of alpha 1,2fucosylated antigens seemed to increase the heat sensitivity in mouse colorectal carcinoma cells.

Plant members of the alpha1-->3/4-fucosyltransferase gene family encode an alpha1-->4-fucosyltransferase, potentially involved in Lewis(a) biosynthesis, and two core alpha1-->3-fucosyltransferases.

Three putative alpha1-->3/4-fucosyltransferase (alpha1-->3/4-FucT) genes have been detected in the Arabidopsis thaliana genome. The products of two of these genes have been identified in vivo as core alpha1-->3-FucTs involved in N-glycosylation. An orthologue of the third gene was isolated from a Beta vulgaris cDNA library. The encoded enzyme efficiently fucosylates Galbeta1-->3GlcNAcbeta1-->3Galbeta1-->4Glc. Analysis of the product by 400 MHz (1)H-nuclear magnetic resonance spectroscopy showed that the product is alpha1-->4-fucosylated at the N-acetylglucosamine residue. In vitro, the recombinant B. vulgaris alpha1-->4-FucT acts efficiently only on neutral type 1 chain-based glycan structures. In plants the enzyme is expected to be involved in Lewis(a) formation on N-linked glycans.

Aberrant nodulation response of Vigna umbellata to a Bradyrhizobium japonicum NodZ mutant and nodulation signals.

The (Brady)rhizobium nodulation gene products synthesize lipo-chitin oligosaccharide (LCO) signal molecules that induce nodule primordia on legume roots. In spot inoculation assays with roots of Vigna umbellata, Bradyrhizobium elkanii LCO and chemically synthesized LCO induced aberrant nodule structures, similar to the activity of these LCOs on Glycine soja (soybean). LCOs containing a pentameric chitin backbone and a reducing-end 2-O-methyl fucosyl moiety were active on V. umbellata. In contrast, the synthetic LCO-IV(C16:0), which has previously been shown to be active on G. soja, was inactive on V. umbellata. A B. japonicum NodZ mutant, which produces LCO without 2-O-methyl fucose at the reducing end, was able to induce nodule structures on both plants. Surprisingly, the individual, purified, LCO molecules produced by this mutant were incapable of inducing nodule formation on V. umbellata roots. However, when applied in combination, the LCOs produced by the NodZ mutant acted cooperatively to produce nodulelike structures on V. umbellata roots.

Purification, cDNA cloning, and expression of GDP-L-Fuc:Asn-linked GlcNAc alpha1,3-fucosyltransferase from mung beans.

Substitution of the asparagine-linked GlcNAc by alpha1,3-linked fucose is a widespread feature of plant as well as of insect glycoproteins, which renders the N-glycan immunogenic. We have purified from mung bean seedlings the GDP-L-Fuc:Asn-linked GlcNAc alpha1,3-fucosyltransferase (core alpha1,3-fucosyltransferase) that is responsible for the synthesis of this linkage. The major isoform had an apparent mass of 54 kDa and isoelectric points ranging from 6. 8 to 8.2. From that protein, four tryptic peptides were isolated and sequenced. Based on an approach involving reverse transcriptase-polymerase chain reaction with degenerate primers and rapid amplification of cDNA ends, core alpha1,3-fucosyltransferase cDNA was cloned from mung bean mRNA. The 2200-base pair cDNA contained an open reading frame of 1530 base pairs that encoded a 510-amino acid protein with a predicted molecular mass of 56.8 kDa. Analysis of cDNA derived from genomic DNA revealed the presence of three introns within the open reading frame. Remarkably, from the four exons, only exon II exhibited significant homology to animal and bacterial alpha1,3/4-fucosyltransferases which, though, are responsible for the biosynthesis of Lewis determinants. The recombinant fucosyltransferase was expressed in Sf21 insect cells using a baculovirus vector. The enzyme acted on glycopeptides having the glycan structures GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1- 6)Manbeta1-4GlcNAcbet a1-4GlcNAcbeta1-Asn, GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1- 6)Manbeta1-4GlcNAcbet a1-4(Fucalpha1-6)GlcNAcbeta1-Asn, and GlcNAcbeta1-2Manalpha1-3[Manalpha1-3(Manalpha1-6 )Manalpha1-6]Manbeta1 -4GlcNAcbeta1-4GlcNAcbeta1-Asn but not on, e.g. N-acetyllactosamine. The structure of the core alpha1,3-fucosylated product was verified by high performance liquid chromatography of the pyridylaminated glycan and by its insensitivity to N-glycosidase F as revealed by matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Purification and cDNA cloning of GDP-L-Fuc:N-acetyl-beta-D-glucosaminide:alpha1-6 fucosyltransferase (alpha1-6 FucT) from human gastric cancer MKN45 cells.

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide:alpha1-6 fucosyltransferase (alpha1-6 FucT), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a culture supernatant of human gastric cancer cell line MKN45. The purification procedures included chromatographies on Q-Sepharose Fast Flow, synthetic GDP-hexanolamine-Sepharose, and GnGn-bi-Asn-Sepharose columns. SDS-PAGE of the purified enzyme gave a major band corresponding to an apparent molecular mass of 60 kDa. The enzyme was recovered in a 12% final yield with an approximately 4,600-fold increase in specific activity. The pH optimum was 7.5, and the enzyme was fully active in the presence of 5 mM EDTA and did not require divalent cations, Mg2+ and Ca2+. Oligonucleotide primers designed from partial amino acid sequences were used to amplify and clone alpha1-6 FucT cDNA from a cDNA library of MKN45 cells. The cDNA encodes 575 amino acids in length, and contains the predicted N-terminal and internal amino acid sequences derived on lysyl endopeptidase digestion. The homology to porcine brain alpha1-6 FucT is 92.2% at the nucleotide level and 95.7% at the amino acid level. No putative N-glycosylation sites were found in the predicted amino acid sequence of the human MKN45 cell enzyme or that of porcine brain. Thus, the enzyme is distinct from other fucosyltransferases which catalyze alpha1-2, alpha1-3, and alpha1-4 fucose addition.

NodZ of Bradyrhizobium extends the nodulation host range of Rhizobium by adding a fucosyl residue to nodulation signals.

The nodulation genes of rhizobia are involved in the production of the lipo-chitin oligosaccharides (LCO), which are signal molecules required for nodule formation. A mutation in nodZ of Bradyrhizobium japonicum results in the synthesis of nodulation signals lacking the wild-type 2-O-methylfucose residue at the reducting-terminal N-acetylglucosamine. This phenotype is correlated with a defective nodulation of siratro (Macroptilium atropurpureum). Here we show that transfer of nodZ to Rhizobium leguminosarum blovar (bv) viciae, which produces LCOs that are not modified at the reducing-terminal N-acetylglucosamine, results in production of LCOs with a fucosyl residue on C-6 of the reducing-terminal N-acetylglucosamine. This finding, together with in vitro enzymatic assays, indicates that the product of nodZ functions as a fucosyltransferase. The transconjugant R. leguminosarum strain producing fucosylated LCOs acquires the capacity to nodulate M. atropurpureum, Glycine soja, Vigna unguiculate and Leucaena leucocephala. Therefore, nodZ extends the narrow host range of R. leguminosarum bv. viciae to include various tropical legumes. However, microscopic analysis of nodules induced on siratro shows that these nodules do not contain bacteroids, showing that transfer of nodZ does not allow R. leguminosarum to engage in a nitrogen-fixing symbiosis with this plant.

Evidence for two distinct alpha(1,2)-fucosyltransferase genes differentially expressed throughout the rat colon.

Blood-group-ABH antigens are carbohydrate structures widely distributed in numerous tissues. These structures are fucosylated by an alpha(1,2)-fucosyltransferase. The occurrence of at least two alpha(1,2)-fucosyltransferase genes in the human genome has been strongly suggested by genetic studies, but only one of them has been cloned so far. Specific primers deduced from this human cDNA were used to amplify a fragment of rat genomic DNA (FTA). Screening of a rat colon cDNA library with this probe allowed us to isolate a clearly distinct, but related, cDNA clone (FTB). Both sequences showed considerable sequence similarity to the human alpha(1,2)-fucosyltransferase cDNA previously cloned. Furthermore, cells transfected with these DNA fragments in antisense orientation displayed a decreased alpha(1,2)-fucosyltransferase activity, indicating that they both correspond to fragments of alpha(1,2)-fucosyltransferase genes. Finally, differential expression of these genes was demonstrated in two rat colon-cancer cell lines and throughout the rat colon.