Francis H. Martin

Stem cell factor is encoded at the SI locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor

We have cloned a partial cDNA encoding murine stem cell factor (SCF) and show that the gene is syntenic with the Sl locus on mouse chromosome 10. Using retroviral vectors to immortalize fetal liver stromal cell lines from mice harboring lethal mutations at the Sl locus (Sl/Sl), we have shown that SCF genomic sequences are deleted in these lines. Furthermore, two other mutations at Sl, Sld and Sl12H, are associated with deletions or alterations of SCF genomic sequences. In vivo administration of SCF can reverse the macrocytic anemia and locally repair the mast cell deficiency of Sl/Sld mice. We have also provided biological and physical evidence that SCF is a ligand for the c-kit receptor.

Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor MpI

A novel megakaryocyte growth and development factor (MGDF) has been identified in aplastic canine plasma, and its cDNAs have been cloned from canine, murine, and human sources. Purified canine MGDF isolated by procedures involving MpI receptor affinity chromatography exists in at least two forms, with apparent molecular masses of 25 kDa and 31 kDa, that share the N-terminal amino acid sequence APP-ACDPRLLNKMLRDSHVLH. Human, dog, and mouse cDNAs for MGDF are highly conserved and encode open reading frames for proteins of 353, 352, and 356 amino acids, respectively, including predicted signal peptides. Canine MGDF and recombinant human MGDF support the development of megakaryocytes from human CD34+ progenitor cell populations in liquid culture and promote the survival of a factor-dependent murine cell line (32D) engineered to express MpI. These biological activities are blocked by the soluble extracellular domain of MpI. These data demonstrate that MGDF is a novel cytokine that regulates megakaryocyte development and is a ligand for the MPI receptor.

Primary structure and functional expression of rat and human stem cell factor DNAs.

Partial cDNA and genomic clones of rat stem cell factor (SCF) have been isolated. Using probes based on the rat sequence, partial and full-length cDNA and genomic clones of human SCF have been isolated. Based on the primary structure of the 164 amino acid protein purified from BRL-3A cells, truncated forms of the rat and human proteins have been expressed in E. coli and mammalian cells and have been shown to possess biological activity. SCF is able to augment the proliferation of both myeloid and lymphoid hematopoietic progenitors in bone marrow cultures. SCF exhibits potent synergistic activities in conjunction with colony-stimulating factors, resulting in increased colony numbers and colony size.

BOTH FAMILIAL PARKINSON'S DISEASE MUTATIONS ACCELERATE ALPHA -SYNUCLEIN AGGREGATION

Parkinson’s disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major component of which are filaments consisting of α-synuclein. Two recently identified point mutations in α-synuclein are the only known genetic causes of PD, but their pathogenic mechanism is not understood. Here we show that both wild type and mutant α-synuclein form insoluble fibrillar aggregates with antiparallel β-sheet structure upon incubation at physiological temperature in vitro. Importantly, aggregate formation is accelerated by both PD-linked mutations. Under the experimental conditions, the lag time for the formation of precipitable aggregates is about 280 h for the wild type protein, 180 h for the A30P mutant, and only 100 h for the A53T mutant protein. These data suggest that the formation of α-synuclein aggregates could be a critical step in PD pathogenesis, which is accelerated by the PD-linked mutations.

Base-base mismatches. Thermodynamics of double helix formation for dCA3XA3G + dCT3YT3G (X, Y = A,C,G,D

Thermodynamic parameters for double strand formation have been measured for the sixteen double helices of the sequence dCA3XA3G.dCT3YT3G, with each of the bases A, C, G and T at the positions labelled X and Y. The results are analyzed in terms of nearest-neighbors and are compared with thermodynamic parameters for RNA secondary structure. At room temperature the sequence (Formula: see text) is more stable than (Formula: see text) and is similar in stability to (Formula: see text) and (Formula: see text) are least stable. At higher temperatures the sequences containing a G.C base pair become more stable than those containing only A.T. All molecules containing mismatches are destabilized with respect to those with only Watson-Crick pairing, but there is a wide range of destabilization. At room temperature the most stable mismatches are those containing guanine (G.T, G.G, G.A); the least stable contain cytosine (C.A, C.C). At higher temperatures pyrimidine-pyrimidine mismatches become the least stable.

Stem cell factor

Stem cell factor (SCF) is the ligand for the tyrosine kinase receptor okit, which is expressed on both primitive and mature hematopoietic progenitor cells. In vitro, SCF synergizes with other growth factors, such as granulocyte colony‐stimulating factor (G–CSF), granulocyte macrophage–colony‐stimulating factor, and interleukin‐3 to stimulate the proliferation and differentiation of cells of the lymphoid, myeloid, erythroid, and megakaryocytic lineages. In vivo, SCF also synergizes with other growth factors and has been shown to enhance the mobilization of peripheral blood progenitor cells in combination with G–CSF. In phase I/II clinical studies administration of the combination of SCF and G‐CSF resulted in a two‐ to threefold increase in cells that express the CD34 antigen compared with G–CSF alone. Other potential clinical uses include ex vivo expansion protocols and in vitro culture for gene therapy. J. Leukoc. Biol. 58: 14–22; 1995.

Parkinson's Disease-associated α-Synuclein Is More Fibrillogenic than β- and γ-Synuclein and Cannot Cross-seed Its Homologs

Parkinsons disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies. Recently, two point mutations in α-synuclein were found to be associated with familial PD, but as of yet no mutations have been described in the homologous genes β- and γ-synuclein. α-Synuclein forms the major fibrillar component of Lewy bodies, but these do not stain for β- or γ-synuclein. This result is very surprising, given the extent of sequence conservation and the high similarity in expression and subcellular localization, in particular between α- and β-synuclein. Here we compare in vitro fibrillogenesis of all three purified synucleins. We show that fresh solutions of α-, β-, and γ- synuclein show the same natively unfolded structure. While over time α-synuclein forms the previously described fibrils, no fibrils could be detected for β- and γ-synuclein under the same conditions. Most importantly, β- and γ-synuclein could not be cross-seeded with α-synuclein fibrils. However, under conditions that drastically accelerate aggregation, γ-synuclein can form fibrils with a lag phase roughly three times longer than α-synuclein. These results indicate that β- and γ-synuclein are intrinsically less fibrillogenic than α-synuclein and cannot form mixed fibrils with α-synuclein, which may explain why they do not appear in the pathological hallmarks of PD, although they are closely related to α-synuclein and are also abundant in brain.

Stem cell factor (SCF), a novel hematopoietic growth factor and ligand for c-kit tyrosine kinase receptor, maps on human chromosome 12 between 12q14.3 and 12qter

Recently a novel hematopoietic growth factor, stem cell factor (SCF), was cloned and demonstrated to be the ligand for the c-kit tyrosine kinase receptor. In the mouse, SCF is encoded bySl (steel), a gene critical to the development of several distinct cell lineages during embryonic life and which has important effects on hematopoiesis in the adult animal. TheS1/SCF locus maps to the distal region of mouse chromosome 10, in the vicinity of genes that have been mapped to human chromosome 12. Here we report the use of somatic cell hybrid lines to localizeSCF to the long arm of human chromosome 12, between 12q14.3 and 12qter. In addition to localizing theSl homolog in man, these data provide further evidence for the conservation of synteny between the long arm of human chromosome 12 and the distal end of mouse chromosome 10.

Contribution of adventitial myofibroblasts to vascular remodeling and lesion formation after experimental angioplasty in pig coronary arteries.

Post-angioplasty restenosis is one of the major problems confronting cardiology today. More than 300,000 angioplasties are performed every year in the United States alone and 3040% of these will be affected by development of a post-angioplasty restenosis lesion. While cellular proliferation after balloon injury is the most likely mechanism for the development of clinical post-angioplasty restenosis, there is controversy regarding the degree of the proliferative response. Endothelial denudation of peripheral vessels using a Fogarty catheter in combination with various degrees of smooth muscle cell damage in rats and rabbits induces smooth muscle cell proliferation and development of a neointima. This has been used extensively as a model for post-angioplasty restenosis. In these models cell proliferation begins in the media within 48 hours after injury. After a week these cells migrate across the internal elastic lamina to form an intimal mass of actively proliferating cell^.^,^ The focus of this work continues to be the regulation of proliferation in the intima or media with very little consideration about how other vascular layers may contribute to the problem. Pigs have been used as models for post-angioplasty restenosis with some success.5-12 Since the coronary arteries of the pig and human are similar in size, the same