Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor (developmental ABSTRACTWe investigated the effects of a newly recognized multifunctional growth factor, the c-kit ligand stem cell factor (SCF), on mouse mast cell proliferation and phenotype.Recombinant rat SCF'64 (rrSCF"64) induced the development of large numbers of dermal mast cells in normal mice in vivo.Many of these mast cells had features of "connective tissue-type mast cells" (CTMC), in that they were reactive both with the heparin-binding fluorescent dye berberine sulfate and with safranin. In vitro, rrSCFI64 induced the proliferation of cloned interleukin 3 (IL-3)-dependent mouse mast cells and primary populations of IL-3-dependent, bone marrow-derived cultured mast cells (BMCMC), which represent immature mast cells, and purified peritoneal mast cells, which represent a type of mature CTMC. BMCMC maintained in rrSCF164 not only proliferated but also matured. Prior to exposure to rrSCF'"4,

A physical map of Arabidopsis thaliana chromosome 4 was constructed in yeast artificial chromosome clones and used to analyze the organization of the chromosome. Mapping of the nucleolar organizing region and the centromere integrated the physical and cytogenetic maps. Detailed comparison of physical with genetic distances showed that the frequency of recombination varied substantially, with relative hot and cold spots occurring along the whole chromosome. Eight repeated DNA sequence families were found in a complex arrangement across the centromeric region and nowhere else on the chromosome.

We recently succeeded in purifying a monocyte chemotactic factor (MCF) from the conditioned media of a myelomonocytic cell line (THP-1) (1). We have molecularly cloned the cDNA of MCF (2), and shown that MCF, in addition to its monocyte chemotactic activity, also activates monocytes to be more cytostatic against several types ofhuman tumor cells in vitro (1) . Therefore, we termed this molecule monocyte chemotactic and activating factor (MCAF). Subsequently, MCAF has been shown to be produced by several additional cell types, namely fibroblasts and endothelial cells (3) . We describe here that MCAF, which was purified from human fibrosarcoma cell line-conditioned media, also induces superoxide anion and lysosomal enzyme release from human monocytes and has potent in vivo monocyte recruitment activity. Materials and MethodsPurification of MCAF. Confluent fibrosarcoma cells, 8387 (4), were stimulated with 100 ng/ml rTNF-a in serum-free media overnight. The culture supernatants were centrifuged, concentrated, and dialyzed against 0.05 M Tris-HCI, pH 7.5, before being applied to a 50-ml heparin Sepharose column . MCAF was eluted with 0.05 M Tris-HCI, pH 7.5, containing 0.5 M NaCl. The eluate was further concentrated and loaded on to a gel filtration column of Sephacryl S-200 (Pharmacia Fine Chemicals, Uppsala, Sweden), which was equilibrated with D-PBS. The fractions containing monocyte chemotaxis activity in the molecular mass range of 10-20 kD were pooled and then applied to a carboxymethyl-silica gel column (7.5 x 150-mm CM-3SW) (Toyo Soda, Tokyo, Japan) connected to an HPLC system (2150 ; LKB Instruments, Inc., Uppsala, Sweden) . The starting buffer was 0.02 M 3-[N-morpholino] propanesulfonic acid (MOPS), pH 6.5, and MCAF was eluted with a linear increase of salt concentration up to 0.5 M NaCl. Finally, samples were applied to a reverse phase chromatography column (4.6 x 750-mm TMS-250 Ultropac column, 10,m; Toyo Soda) with the starting

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In order to analyse further the genomic distribution of repetitive sequences in the Arabidopsis genome, we have identified and characterized seven novel repetitive sequences. Analysis of genomic representation, genomic location and DNA sequence divided the seven repeated sequences into two classes. The first was represented by three cosmid subclones (182A, 74A, 191A) carrying sequences that hybridised to up to 20 genomic fragments and showed sequence homology to the genes, Arabidopsis CCR2, Arabidopsis MYB and to various ATP-binding transport proteins. These multigene families mapped to various positions within the genome, as judged by hybridization to YAC clones constituting the Arabidopsis physical map. The second class was represented by four cosmid subclones (106B, 164A, 163A, 278A) that hybridised to between 20 and 300 genomic fragments. One of these, 106B, is a diverged, partial copy of the LTR of the Arabidopsis retrotransposon Athila. The other three sequences showed no homology to known genes or proteins. The distribution of these sequences on chromosome 4 was analysed and sequences hybridizing to 106B, 164A and 163A were found exclusively at the centromeric region of this chromosome. Their detailed arrangement at the centromeric region of chromosome 4, relative to other repeated sequence families and single copy sequences, was determined.

Incubation of human polymorphonuclear leucocytes (PMN) with either the chemotactic factor N-formylmethionyl-leucylphenylalanine (FMLP) or phorbol 12-myristate 13-acetate (PMA) activates a kinase with phosphorylating activity towards a known microtubule-associated protein-2 (MAP) kinase substrate, the epidermal growth factor receptor peptide (T669). Activation of this enzyme by FMLP was maximal at 1 min, decreasing by 10 min. Activation by PMA was slightly slower than that by FMLP, but more prolonged (maximal at 5 min, with no significant decrease by 20 min). The enzyme induced by either stimulant bound strongly to phenyl-Sepharose, had a molecular mass of 40 kDa on gel filtration and phosphorylated three MAP kinase substrates, i.e. MAP, myelin basic protein and the T669 peptide. By use of antibodies to MAP kinases and phosphotyrosine, the enzyme was identified as the 42 kDa MAP kinase (also known as extracellular-signal-regulated kinase 2, ERK2). Stimulation of PMN with FMLP or PMA was also found to induce a kinase kinase which phosphorylated human recombinant MAP kinase on threonine and tyrosine, with concomitant activation. These results suggest that MAP kinase and the kinase kinase are involved in the activation of PMN by chemotactic factors such as FMLP.

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Mast cells are resident in tissues, particularly in association with endothelial and epithelial cell basement membranes, and increase at sites of inflammation, injury, and fibrosis. Although mast cells are known to both release and generate proinflammatory molecules in response to inflammatory stimuli, little is known about their normal biologic function. Here we demonstrate that IL-3-dependent mouse PT18 mast cells, mouse bone marrow-derived mast cells, and rat basophilic leukemia cells express large amounts of mRNA for collagen IV, laminin, and heparan sulfate proteoglycan. Western blot analysis confirmed that mast cells synthesize and secrete significant amounts collagen IV and laminin B1 and B2 chains. These data suggest that mast cells may contribute to normal tissue repair and/or the early overproduction ofbasement membrane components seen in a variety of fibrotic conditions. (J. Clin. Invest.