Trafficking and localization studies of recombinant α1,3-fucosyltransferase VI stably expressed in CHO cells

Peripheral α1,3-fucosylation of glycans occurs by the action of either one of five different α1,3-fucosyltransferases (Fuc-Ts) cloned to date. Fuc-TVI is one of the α1,3-fucosyltransferases which is capable to synthesize selectin ligands. The major α1,3-fucosyltransferase activity in human plasma is encoded by the gene for fucosyltransferase VI, which presumably originates from liver cells. While the sequence, chromosomal localization, and kinetic properties of Fuc-TVI are known, immunocytochemical localization and trafficking studies have been impossible because of the lack of specific antibodies. Here we report on the development and characterization of a peptide-specific polyclonal antiserum mono-specific to Fuc-TVI and an antiserum to purified soluble recombinant Fuc-TVI crossreactive with Fuc-TIII and Fuc-TV. Both antisera were applied for immunodetection in stably transfected CHO cells expressing the full-length form of this enzyme (CHO clone 61/11). Fuc-TVI was found to be a resident protein of the Golgi apparatus. In addition, more than 30% of cell-associated and released enzyme activity was found in the medium. Maturation and release of Fuc-TVI was analyzed in metabolically labeled CHO 61/11 cells followed by immunoprecipitation. Fuc-TVI occurred in two forms of 47 kDa and 43 kDa bands, while the secreted form was detected as a 43 kDa. These two different intracellular forms arose by posttranslational modification, as shown by pulse-chase experiments. Fuc-TVI was released to the supernatant by proteolytic cleavage as a partially endo-H resistant glycofor

Nuclear orphan receptor NR2F6 directly antagonizes NFAT and RORγt binding to the Il17a promoter

AbstractInterleukin-17A (IL-17A) is the signature cytokine produced by Th17 CD4+ T cells and has been tightly linked to autoimmune pathogenesis. In particular, the transcription factors NFAT and RORγt are known to activate Il17a transcription, although the detailed mechanism of action remains incompletely understood. Here, we show that the nuclear orphan receptor NR2F6 can attenuate the capacity of NFAT to bind to critical regions of the Il17a gene promoter. In addition, because NR2F6 binds to defined hormone response elements (HREs) within the Il17a locus, it interferes with the ability of RORγt to access the DNA. Consistently, NFAT and RORγt binding within the Il17a locus were enhanced in Nr2f6-deficient CD4+ Th17 cells but decreased in Nr2f6-overexpressing transgenic CD4+ Th17 cells. Taken together, our findings uncover an example of antagonistic regulation of Il17a transcription through the direct reciprocal actions of NR2F6 versus NFAT and RORγt

Mechanistic studies on the epoxidation of P Oleovorans

Ph.D.Sheldon W. Ma

Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory signals. Rolling under the hydrodynamic drag forces of blood flow is mediated by the interaction between selectins and their ligands across the leukocyte and endothelial cell surfaces. Here we present force-spectroscopy experiments on single complexes of P-selectin and P-selectin glycoprotein ligand-1 by atomic force microscopy to determine the intrinsic molecular properties of this dynamic adhesion process. By modeling intermolecular and intramolecular forces as well as the adhesion probability in atomic force microscopy experiments we gain information on rupture forces, elasticity, and kinetics of the P-selectin/P-selectin glycoprotein ligand-1 interaction. The complexes are able to withstand forces up to 165 pN and show a chain-like elasticity with a molecular spring constant of 5.3 pN nm(−1) and a persistence length of 0.35 nm. The dissociation constant (off-rate) varies over three orders of magnitude from 0.02 s(−1) under zero force up to 15 s(−1) under external applied forces. Rupture force and lifetime of the complexes are not constant, but directly depend on the applied force per unit time, which is a product of the intrinsic molecular elasticity and the external pulling velocity. The high strength of binding combined with force-dependent rate constants and high molecular elasticity are tailored to support physiological leukocyte rolling

Non-covalent polyvalent ligands by self-assembly of small glycodendrimers: a novel concept for the inhibition of polyvalent carbohydrate-protein interactions in vitro and in vivo.

Polyvalent carbohydrate-protein interactions occur frequently in biology, particularly in recognition events on cellular membranes. Collectively, they can be much stronger than corresponding monovalent interactions, rendering it difficult to control them with individual small molecules. Artificial macromolecules have been used as polyvalent ligands to inhibit polyvalent processes; however, both reproducible synthesis and appropriate characterization of such complex entities is demanding. Herein, we present an alternative concept avoiding conventional macromolecules. Small glycodendrimers which fulfill single molecule entity criteria self-assemble to form non-covalent nanoparticles. These particles-not the individual molecules-function as polyvalent ligands, efficiently inhibiting polyvalent processes both in vitro and in vivo. The synthesis and characterization of these glycodendrimers is described in detail. Furthermore, we report on the characterization of the non-covalent nanoparticles formed and on their biological evaluation

Combinations of Anti-LFA-1, Everolimus, Anti-CD40 Ligand, and Allogeneic Bone Marrow Induce Central Transplantation Tolerance through Hemopoietic Chimerism, Including Protection from Chronic Heart Allograft Rejection

Central transplantation tolerance through hemopoietic chimerism initially requires inhibition of allogeneic stem cell or bone marrow (BM) rejection, as previously achieved in murine models by combinations of T cell costimulation blockade. We have evaluated LFA-1 blockade as part of regimens to support mixed hemopoietic chimerism development upon fully allogeneic BALB/c BM transfer to nonirradiated busulfan-treated B6 recipient mice. Combining anti-LFA-1 with anti-CD40 ligand (CD40L) induced high incidences and levels of stable multilineage hemopoietic chimerism comparable to chimerism achieved with anti-CD40L and everolimus (40-O-(2-hydroxyethyl)-rapamycin) under conditions where neither Ab alone was effective. The combination of anti-LFA-1 with everolimus also resulted in high levels of chimerism, albeit with a lower incidence of stability. Inhibition of acute allograft rejection critically depended on chimerism stability, even if maintained at very low levels around 1%, as was the case for some recipients without busulfan conditioning. Chimerism stability correlated with a significant donor BM-dependent loss of host-derived Vbeta11(+) T cells 3 mo after BM transplantation (Tx). Combinations of anti-CD40L with anti-LFA-1 or everolimus also prevented acute rejection of skin allografts transplanted before established chimerism, albeit not independently of allospecific BMTx. All skin and heart allografts transplanted to stable chimeras 3 and 5 mo after BMTx, respectively, were protected from acute rejection. Moreover, this included prevention of heart allograft vascular intimal thickening ("chronic rejection")

Combinations of anti-LFA-1, everolimus, anti-CD40 ligand, and allogeneic bone marrow induce central transplantation tolerance through hemopoietic chimerism, including protection from chronic heart allograft rejection.

Central transplantation tolerance through hemopoietic chimerism initially requires inhibition of allogeneic stem cell or bone marrow (BM) rejection, as previously achieved in murine models by combinations of T cell costimulation blockade. We have evaluated LFA-1 blockade as part of regimens to support mixed hemopoietic chimerism development upon fully allogeneic BALB/c BM transfer to nonirradiated busulfan-treated B6 recipient mice. Combining anti-LFA-1 with anti-CD40 ligand (CD40L) induced high incidences and levels of stable multilineage hemopoietic chimerism comparable to chimerism achieved with anti-CD40L and everolimus (40-O-(2-hydroxyethyl)-rapamycin) under conditions where neither Ab alone was effective. The combination of anti-LFA-1 with everolimus also resulted in high levels of chimerism, albeit with a lower incidence of stability. Inhibition of acute allograft rejection critically depended on chimerism stability, even if maintained at very low levels around 1%, as was the case for some recipients without busulfan conditioning. Chimerism stability correlated with a significant donor BM-dependent loss of host-derived Vbeta11(+) T cells 3 mo after BM transplantation (Tx). Combinations of anti-CD40L with anti-LFA-1 or everolimus also prevented acute rejection of skin allografts transplanted before established chimerism, albeit not independently of allospecific BMTx. All skin and heart allografts transplanted to stable chimeras 3 and 5 mo after BMTx, respectively, were protected from acute rejection. Moreover, this included prevention of heart allograft vascular intimal thickening ("chronic rejection")

In Silico and In Vitro Analysis of lncRNA XIST Reveals a Panel of Possible Lung Cancer Regulators and a Five-Gene Diagnostic Signature

Long non-coding RNAs (lncRNAs) perform a wide functional repertoire of roles in cell biology, ranging from RNA editing to gene regulation, as well as tumour genesis and tumour progression. The lncRNA X-inactive specific transcript (XIST) is involved in the aetiopathogenesis of non-small cell lung cancer (NSCLC). However, its role at the molecular level is not fully elucidated. The expression of XIST and co-regulated genes TSIX, hnRNPu, Bcl-2, and BRCA1 analyses in lung cancer (LC) and controls were performed in silico. Differentially expressed genes (DEGs) were determined using RNA-seq in H1975 and A549 NSCLC cell lines following siRNA for XIST. XIST exhibited sexual dimorphism, being up-regulated in females compared to males in both control and LC patient cohorts. RNA-seq revealed 944 and 751 DEGs for A549 and H1975 cell lines, respectively. These DEGs are involved in signal transduction, cell communication, energy pathways, and nucleic acid metabolism. XIST expression associated with TSIX, hnRNPu, Bcl-2, and BRCA1 provided a strong collective feature to discriminate between controls and LC, implying a diagnostic potential. There is a much more complex role for XIST in lung cancer. Further studies should concentrate on sex-specific changes and investigate the signalling pathways of the DEGs following silencing of this lncRNA

Modulation of T cell homeostasis and alloreactivity under continuous FTY720 exposure.

The immunomodulator FTY720 inhibits lymph node (LN) and thymic egress, thereby constraining T cell circulation and reducing peripheral T cell numbers. Here, we analyzed in mouse models the as yet scarcely characterized impact of long-term (up to 6 months) FTY720 exposure on T cell homeostasis and possible consequences for alloreactivity. In green fluorescent protein (GFP) hemopoietic chimeras, the turnover of (initially GFP(-)) peripheral T cell pools was markedly delayed under FTY720, while normal homeostatic differences between CD4 and CD8 T cell sub-populations were retained or amplified further. Homeostatic proliferation was enhanced, and within shrinking T cell pools, the proportions of effector memory phenotype CD4 T cells (CD4T(PEM)) increased in spleens and LNs and of central memory phenotype CD8 T cells (CD8T(PCM)) in LNs. By contrast, the fractions of CD8T(PEM) and CD4T(PCM) remained stably small under FTY720. The enrichment for CD4T(PEM) and CD8T(PCM) correlated with larger proportions of IFNgamma-producing T cells upon nonspecific but not allospecific stimulation. Splenic CD4 T cells from FTY720-treated mice proliferated more strongly upon transfer to semi-allogeneic hosts. However, heart allograft survival was not compromised in FTY720 pre-treated recipients. It correlated with reduced intra-graft CD8 T cells, and the longest surviving transplants contained the highest numbers of CD4 T cells. Thus, continuous FTY720 exposure reveals differential homeostatic responses by memory phenotype CD4 and CD8 T cell sub-populations, and it may enhance alloreactive CD4 T cell proliferation and tissue infiltration without accelerating allograft rejection