Human hematopoietic cell transplantation results in generation of donor-derived epithelial cells.
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Epithelial Cells↗
Biomedical subjects
M Follo
Publications and source records attributed to M Follo.
The formin-binding protein 17, FBP17, binds via a TNKS binding motif to tankyrase, a protein involved in telomere maintenance.
In acute myelogenous and lymphoid leukemias, rearrangements involving the MLL (mixed lineage leukemia) gene at chromosome 11q23 are frequent. The truncated MLL protein is fused in-frame to a series of partner proteins. We previously identified the formin-binding protein 17 (FBP17) as such an MLL fusion partner. In this study, we explored in vivo physiological interaction partners of FBP17 using a two-hybrid assay and found tankyrase (TNKS), an ADP-ribose polymerase protein involved in telomere maintenance and mitogen-activated protein kinase signaling. We demonstrate that FBP17 binds via a special TNKS-binding motif to tankyrase. The physiological relevance is indicated by co-immunoprecipitation of endogenous proteins in 293T cells.
Amino Acid Motifs↗
Expansion and fibronectin-enhanced retroviral transduction of primary human T lymphocytes for adoptive immunotherapy.
Human lymphocytes remain among the most promising target cells for gene therapy. Gene-modified lymphocytes have been used successfully to treat adenosine deaminase (ADA)-deficient patients and to control GvHD after allogeneic BMT. Because activation and proliferation of T cells are necessary for efficient retrovirus-mediated gene transfer and subsequent selection of transduced cells, mononuclear cells (MNC) from steady-state and G-CSF-stimulated peripheral blood were activated by short exposure to the mitogen PHA, the anti-CD3 antibody OKT3, or both in the presence of different concentrations of recombinant IL-2. Using OKT3 (10 or 30 ng/ml) and IL-2 (100 U/ml), T cells expanded efficiently during a 14-day culture period. Cell expansion was similar under serum-free conditions. The immunophenotypic profile over time showed a marked increase in CD8+ cells, leading to a reversed CD4/CD8 ratio of 1:2 and a slight increase in CD56+ cells. Supernatant-based centrifugal transduction of primary human T lymphocytes was compared with supernatant transduction on the extracellular matrix protein fibronectin. Transduction with cell-free retrovirus-containing supernatant in tissue culture flasks coated with human plasma fibronectin led to significantly higher transduction efficiencies (20% +/- 7.5%) than centrifugal transduction in uncoated culture flasks (13.6% +/- 5.1%)(p = 0.041). To both rapidly characterize transduced cells and isolate these from residual nontransduced but biologically equivalent cells, an amphotropic Moloney murine leukemia virus (MoMuLV)-based retroviral vector containing the intracytoplasmically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene was used. FACS sorting of T cells after transduction resulted in >90% LNGFR+ cells and was much faster than enrichment of transduced cells through growth in G418-selection medium. These results show that supernatant-based retroviral gene transfer into primary human T lymphocytes can be enhanced by fibronectin. Ectopic expression of a cell surface protein can be used to rapidly and conveniently quantitate transduction efficiency through FACS analysis and to efficiently enrich transduced cells through FACS sorting.
Gene Transfer Techniques↗
Identification of interaction partners for the basic-helix-loop-helix protein E47.
Helix-loop-helix proteins constitute a family of transcription factors with the potential to form homo- and hetero-dimers mediated by the helix-loop-helix domain. Oncogenic mutations in such genes can disrupt the equilibrium of protein-protein interactions in the affected cell. In order to assess the biological consequences of such mutations, the full complement of interacting proteins must be known. To identify proteins interacting with the basic-helix-loop-helix domain of the ubiquitously expressed E47 protein, a 'sandwich'-screening procedure was developed which distinguishes between homo- and hetero-oligomers, and specifically excludes the detection of complexes which cannot bind DNA. Nine distinct cDNAs were identified which encode proteins with apparent basic-helix-loop-helix domains, including a novel clone termed eip1 which is distantly related in the basic-helix-loop-helix domain to the Drosophila enhancer-of-split m7 protein. Using epitope-tagging, interaction of E47 basic-helix-loop-helix protein with the eip1 protein encoded by this novel cDNA was confirmed by immunoprecipitation experiments in COS7 cells. Interaction was also observed in the yeast two-hybrid system. Three cDNAs encoding proteins without basic-helix-loop-helix domains were also found to interact in the sandwich-expression screen. Interactions with human PARP and mouse replication factor 1a were confirmed using glutathione transferase-tagged cDNAs. A cDNA encoding part of the nucleolin protein sequence interacted with the E47 basic-helix-loop-helix only when fused to a beta-galactosidase tag.
Amino Acid Sequence↗
Dynamic changes in gene expression during in vitro differentiation of mouse embryonic stem cells.
The expression pattern of protein tyrosine kinases (PTK) and phosphatases (PTP) was determined during the first eight days of in vitro differentiation of mouse embryonic stem (ES) cells. DNA fingerprinting of catalytic domains amplified from cDNA revealed dynamic changes in expression of previously described genes. A novel PTP is expressed in undifferentiated ES cells, and is down-regulated during in vitro differentiation.
Animals↗
Structure and expression of the human gene encoding plasminogen activator inhibitor, PAI-1.
The gene (pai1) encoding human plasminogen activator inhibitor, PAI-1 was cloned from a lambda EMBL3 genomic library and was found to span approx. 12 kb and to contain eight introns. Primer extension and S1 nuclease analyses both showed the transcription start point to be located 142 nt upstream from the start codon. The 5'-flanking region was sequenced and found to contain a TATA box, but no CAAT sequence. When a fragment containing 730 nt of 5'-untranslated region was placed upstream from a promoterless cat gene, it was shown to function as a promoter when transfected into COS cells. Northern-blot analysis was consistent with low level expression of the endogenous pai1 gene in COS cells. When the pai1 gene structure was compared to those of other members of the serine-protease-inhibitor encoding gene family, little conservation of intron positions was observed.
Amino Acid Sequence↗