The behavioural and electrophysiological effects of visual task difficulty and bimanual coordination mode during dual-task performance.

The difficulty of a visual three stimulus and a bimanual coordination task was manipulated by varying discrimination difficulty (easy, hard) and coordination mode (in-phase, anti-phase) respectively. Electroencephalographic activity was recorded from 32 sites whilst participants (n = 16) completed four dual-task conditions in counterbalanced order. Longer reaction time and lower accuracy were found for the hard relative to the easy visual task and, for the hard visual task, accuracy was lower under anti-phase relative to in-phase conditions. Amplitude and latency of event-related potential components P3a and P3b were recorded and measured. There was a reduction in P3b amplitude and increase in P3a amplitude for the hard visual task overall and a further reduction in frontal P3b amplitude under the more demanding anti-phase condition. For the easy visual task, however, P3b and P3a amplitude were greater under the anti-phase relative to in-phase coordination condition at left hemisphere frontal sites. These findings suggest that the attentional cost of stabilising anti-phase bimanual coordination is largely associated with top-down automatic processes subserved by the frontal attentional network.

Structure and expression of a novel member, FGF-16, on the fibroblast growth factor family.

We have isolated cDNA encoding a novel member (207 amino acids) of the FGF family from the rat heart by homology-based polymerase chain reaction. As this protein is the 16th documented member of the FGF family, we tentatively term it FGF-16. Among FGF family members, FGF-16 is most similar (73% amino acid identity) to FGF-9. We have also determined the structure of human FGF-16 with high amino acid sequence identity (98.6%) to rat FGF-16. Although the predicted FGF-16 amino acid sequence lacks a typical signal sequence, recombinant rat FGF-16 was efficiently secreted by Sf9 insect cells infected with recombinant baculovirus containing the cDNA. FGF-16 mRNA was predominantly expressed in the rat heart among the adult major tissues examined. The expression profile of FGF-16 mRNA was quite different from those of other members of the FGF family. In rat embryos, FGF-16 mRNA was predominantly expressed in the brown adipose tissue. However, the expression decreased greatly after birth. These results indicate that FGF-16 in embryos might play a role in development of the brown adipose tissue.

Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries.

BACKGROUND: In the present series of experiments, we examined the onset of cell proliferation and growth factor expression after balloon overstretch injury to porcine coronary arteries. METHODS AND RESULTS: Domestic juvenile swine underwent balloon overstretch injury to the left anterior descending and circumflex coronary arteries with standard percutaneous transluminal coronary angioplasty balloon catheters. To identify proliferating cells, 5-bromo-2-deoxyuridine (BrDU) was administered over a period of 24 hours before the animals were killed at either 1, 3, 7, or 14 days after injury. Immunohistochemistry was performed with monoclonal antibodies to BrDU and smooth muscle cell markers. Three days after injury, a large number of proliferating cells were located in the adventitia, with significantly fewer positive cells found in the media and lumen. Seven days after injury, proliferating cells were found primarily in the neointima, extending along the luminal surface. In situ hybridization for PDGF A-chain and beta-receptor mRNAs revealed that the expression of these two genes was closely correlated with the sites of proliferation at each time point. Studies in which BrDU was injected between days 2 and 3 and the animals were killed on day 14 suggested that the proliferating adventitial cells may migrate into the neointima. CONCLUSIONS: These data suggest that adventitial myofibroblasts contribute to the process of vascular lesion formation by proliferating, synthesizing growth factors, and possibly migrating into the neointima. Increased synthesis of alpha-smooth muscle actin observed in the adventitial cells after arterial injury may constrict the injured vessel and contribute to the process of arterial remodeling and late lumen loss after angioplasty.

Avian stem cell factor (SCF): production and characterization of the recombinant His-tagged SCF of chicken and its neutralizing antibody.

Recombinant chicken stem cell factor (SCF) was produced in bacteria as a histidine-tagged protein (His delta SCF) and purified by affinity chromatography on an Ni(2+)-NTA agarose column. His delta SCF is devoid of signal peptide and transmembrane domains and therefore mimics a soluble form of SCF. We demonstrate that the factor is biologically fully active in supporting sustained proliferation of SCF-dependent red blood progenitor cells of chicken in vitro. Polyclonal SCF-specific antibodies were raised in rabbits and shown to display potent SCF-neutralizing activity. Thus, recombinant chicken His delta SCF together with the SCF-specific antibody provide valuable tools for studying the role of SCF and c-kit receptor in development, growth and differentiation of avian haematopoietic cells.

Stem cell factor production by human marrow stromal fibroblasts.

To characterize the production of stem cell factor (SCF, the ligand for the c-kit receptor protein) and its regulation by inflammatory cytokines and glucocorticoids, primary marrow stromal fibroblasts were isolated from normal individuals and two patients with Diamond-Blackfan anemia. Unstimulated normal marrow stromal fibroblasts constitutively expressed a low level of SCF mRNA (9 +/- 2 copies/cell [mean +/- SEM]), continually secreted soluble SCF into the supernatant of 1- to 5-day-old cultures (0.16 +/- 0.02 to 0.73 +/- 0.04 ng/mL per 10(6) cells, respectively), and expressed membrane-bound SCF. Stimulation with interleukin-1 beta (IL-1 beta) only modestly increased SCF mRNA levels, soluble SCF production at 24 hours, and membrane-bound SCF. In comparison, hydrocortisone or tumor necrosis factor alpha (TNF-alpha) exposure increased SCF mRNA levels 3.5- to four-fold above controls, but with different kinetics. The peak TNF-alpha effect was at 6 hours, with return to near control levels at 24 hours, whereas hydrocortisone induced maximal mRNA increases at 12 to 18 hours, and the levels remained high at 24 hours. Similarly, a sustained increase in soluble SCF production was detected during 1 to 5 days of hydrocortisone exposure (0.27 +/- 0.03 to 1.10 +/- 0.08 ng/mL per 10(6) cells), while TNF-alpha stimulation modestly increased the production of soluble SCF in 24-hour cultures only. Unstimulated normal marrow fibroblasts expressed predominantly the long species of alternatively spliced SCF mRNA, and the relative amounts of long and short mRNAs did not change after stimulation with IL-1 beta, hydrocortisone, or TNF-alpha. SCF production by marrow stromal fibroblasts from a symptomatic patient with Diamond-Blackfan anemia was equivalent to simultaneously studied normal marrow fibroblasts. In contrast, marrow fibroblasts from a Diamond-Blackfan anemia patient in untreated hematologic remission constitutively expressed high levels of SCF mRNA (21 +/- 4 copies/cell) and soluble protein (0.40 ng/mL per 10(6) cells at 24 hours). Together, these observations suggest that SCF is constitutively produced by fibroblasts in the human marrow microenvironment and that hydrocortisone induces a modest but sustained increase in SCF gene expression and protein production, compared to only a transient increase induced by TNF-alpha. In addition, these findings support the hypothesis that endogenous or corticosteroid-induced increases in the production of SCF could play a physiologic role in the clinical improvement of congenital anemia.

Steel and c-kit in the development of avian melanocytes: a study of normally pigmented birds and of the hyperpigmented mutant silky fowl.

We describe here the expression of c-kit and Steel (Sl) genes during the development of melanocytes in normally pigmented strains of chick and quail compared to unpigmented (White Leghorn) and hyperpigmented (Silky Fowl) strains of chickens. By using the quail/chick chimera system, we found that the neural crest cells, which migrate dorso-laterally in the subectodermal mesenchyme to give rise to the melanocytes, express c-kit as early as E4, that is about 2 days after they have left the neural primordium. The Sl gene is expressed from E4 onward in the epidermis but not at all in the dermis at any developmental stage. As feather buds develop, Sl mRNA becomes restricted to the apical region of the feather filaments. During formation of the barbs and barbules of the down feather, production of the Steel factor is restricted to the external epidermal cells of the barbules. The cell bodies of the c-kit-positive melanocytes are then located in the internal border of the epidermal ridges and extend their processes toward the source of the Steel factor. We propose that the spatial restriction of Sl gene activity at that stage accounts for the morphology of the melanocytes and their vectorial secretion of melanin to the external barbule cells. As a whole, these results show that during skin development c-kit positive cells are present in the Steel factor-producing areas at the time when melanoblasts proliferate and differentiate. Interestingly, in the mouse, previous studies showed that the Sl gene is activated in the dermis where melanoblasts undergo most of their expansion (Nishikawa et al. [1991] EMBO J. 10:2111-2118). In the unpigmented and hyperpigmented mutants that we studied, expression of the Sl message, as judged quantitatively in Northern blots (for the SF embryos) or spatially by in situ hybridization, is similar to that observed in normal birds. In SF embryos the c-kit expressing melanoblasts migrate initially in the dorso-lateral migration pathway as in normal birds. However their number increases considerably in the dermis from E5 onward. From E7, they invade mesodermally derived organs that do not express the Sl gene. This suggests that another, still unknown, factor(s) is responsible for the survival, the proliferation, and the extensive spreading of melanocytic cells within the mesoderm of this mutant.

Effect of the Steel gene product on melanogenesis in avian neural crest cell cultures.

Mutations at the Steel (Sl) and dominant white spotting (W) loci affect three embryonic lineages: primordial germ cells, hemopoietic stem cells and neural-crest-derived melanocytes. The gene products of these loci are a peptide growth factor, called here stem cell factor (SCF), and its tyrosine kinase receptor, the proto-oncogene c-kit. We have studied how chicken recombinant SCF affects the development of melanocytes from quail neural crest cells in secondary culture under defined conditions. We observed that the total number of neural crest cells, of melanocytes and of their precursors was higher in the presence than in the absence of SCF. Labelling with bromodeoxyuridine showed that SCF had a modest and transient mitogenic effect on the neural crest population. SCF also enhanced the differentiation rate of melanocyte precursors, recognized by the "melanocyte early marker" monoclonal antibody (MelEM MAb), and of melanocytes, since the proportion of both subpopulations significantly increased in the presence of SCF. Finally, SCF increased the survival of the neural crest population since in its presence the total number of cells remained stable while it gradually declined in control cultures. Our results support the notion that SCF sustains the survival of the neural crest population and stimulates the rate of the melanogenic differentiation process.

Canine stem cell factor (c-kit ligand) supports the survival of hematopoietic progenitors in long-term canine marrow culture.

The cDNA for canine stem cell factor (cSCF, c-kit ligand) was cloned and expressed in Escherichia coli. The recombinant protein (rcSCF), 165 amino acids in length, is very similar structurally to the soluble form of previously cloned and sequenced rodent and human SCFs. The biological effects of rcSCF were studied in a day-10 granulocyte-macrophage colony-forming unit (CFU-GM) clonogenic assay and in long-term liquid bone marrow culture of non-adherent hematopoietic cells in the absence of a stromal underlayer. Synergism in the stimulation of growth of CFU-GM was demonstrated between rcSCF and both recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and naturally occurring colony-stimulating activity present in the serum of a neutropenic dog. Alone, rcSCF was nonstimulatory for committed marrow precursors in methylcellulose cultures and had minimal effect on hematopoietic progenitor cell survival in stromaless, liquid cultures. When rcSCF was combined with phytohemagglutinin-stimulated canine lymphocyte-conditioned medium (PHA-LCM) or rh interleukin 6 (IL-6), with or without rhGM-CSF, CFU-GM survived for up to 5 weeks. The combination of rcSCF and rhGM-CSF, without rhIL-6, led to an early increase in CFU-GM in liquid cultures that declined more rapidly than in flasks that included rhIL-6. Survival of progenitor cells was negligible beyond 1 week in flasks with growth factor combinations lacking rcSCF. Sustained production of nonadherent cells in long-term cultures also was dependent on rcSCF in combination with canine PHA-LCM or recombinant human growth factors. It appears that rcSCF, like that from rodent and primate species, has the ability to influence the survival and proliferation of CFU-GM, and perhaps earlier progenitor cells, in hematopoietic tissues. In a long-term liquid culture system in which growth factor production by stromal cells is limited, rcSCF possesses a unique ability to maintain the viability of progenitor cells for up to 5 weeks.

Support of human hematopoiesis in long-term bone marrow cultures by murine stromal cells selectively expressing the membrane-bound and secreted forms of the human homolog of the steel gene product, stem cell factor.

The maintenance and differentiation of hematopoietic stem cells is influenced by cells making up the hematopoietic microenvironment (HM), including bone marrow-derived stromal cells. We and several other investigators have recently demonstrated the molecular basis of abnormal HM observed in the steel mutant mouse and cloned the normal cDNA products of this gene (termed SCF, KL, or MCF). In this report, we focus on the human counterpart of the mouse Steel (Sl) gene. Alternative splicing of the human SCF pre-mRNA transcript results in secreted and membrane-bound forms of the protein. To investigate the role of these two forms of human SCF, we targeted an immortalized stromal cell line derived from fetal murine homozygous (Sl/Sl) SCF-deficient embryos for gene transfer of various human cDNAs encoding SCF. We report that stable stromal cell transfectants can differentially process the two forms of human SCF protein product. We also demonstrate that both soluble SCF and membrane-bound SCF are active in increasing the number of human progenitor cells in the context of stromal cell cultures, although in a qualitatively different manner. Hence, the membrane-bound form of SCF may play an important role in the cell-cell interactions observed between stromal and hematopoietic cells both in vitro and in vivo.

Absence of abnormalities of c-kit or its ligand in two patients with Diamond-Blackfan anemia.

As Diamond-Blackfan anemia shares clinical features with W and Steel defects in mice, we investigated the possibility that this human disorder might result from an abnormality of the c-kit receptor or its ligand, stem cell factor (SCF). For these studies, full nucleotide sequences for coding regions of c-kit and SCF were generated for two Diamond-Blackfan anemia patients and were normal. Similarly, the kds of SCF receptors on their marrow cells (31 pmol/L, 43 pmol/L) were comparable with those found in three normal controls (50 pmol/L, 55 pmol/L, 27 pmol/L). Serum SCF concentrations were 6.9 ng/mL in patient A, 14.6 ng/mL in patient B, who has been in hematologic remission since adolescence, and 2.7 ng/mL in the 3-year-old daughter of patient B, who also has Diamond-Blackfan anemia but is transfusion-dependent. It is possible that the SCF level in patient B increased with puberty, leading to her remission. These data provide evidence that Diamond-Blackfan anemia does not result from structural abnormalities of c-kit or SCF.

Diamond-blackfan anemia: in vitro response of erythroid progenitors to the ligand for c-kit.

To provide insights into the pathogenesis of Diamond-Blackfan anemia, we examined the in vitro response of erythroid progenitors to the recently isolated ligand for c-kit (stem cell factor, SCF). For these studies, marrow or blood mononuclear cells from 10 Diamond-Blackfan patients were cultured with erythropoietin (Ep), Ep and interleukin-3, Ep and granulocyte-macrophage colony-stimulating factor, or Ep and lymphocyte conditioned media (LCM). These combinations were tested in the presence or absence of SCF. The mean number of cells per erythroid burst increased 5 to 50-fold in cultures containing SCF. Furthermore, many additional erythroid bursts were seen (mean increment 3.2 x baseline values). Although burst-forming unit-erythroid (BFU-E) from all patients responded, there were differences among individuals in the sensitivity of their BFU-E to SCF. In six patients and all control studies, plateau frequencies of erythroid bursts were achieved with less than or equal to 10 ng/mL SCF, whereas in studies from the other four patients, over 50 ng/mL SCF was required. These data invite speculation that the c-kit receptor/ligand axis is involved in the pathogenesis of Diamond-Blackfan anemia. More importantly and regardless of whether the observed patterns of response reflect the primary defect or an epiphenomenon, our data strongly support a therapeutic trial of SCF in patients with Diamond-Blackfan anemia.

Platelet factor 4 mRNA expression in human erythroleukemic cells: regulation by phorbol esters and certain cytokines.

Developing megakaryocytes are distinguished from progenitor cells by the appearance of platelet proteins such as platelet factor 4 (PF 4). The human erythroleukemic cell line HEL can also be induced to produce PF 4 by incubation in phorbol esters. HEL cells were used here as a model system in which to study the phenomenon of inducible PF 4 production at both the mRNA and protein levels. The cytokines interleukin 1 beta (IL-1 beta), interleukin 3 (IL-3), interleukin 6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO), and transforming growth factor-beta (TGF-beta) were also evaluated for their effects on PF 4 mRNA induction in HEL cells.

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 by Sl (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. The Sl/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 localize SCF to the long arm of human chromosome 12, between 12q14.3 and 12qter. In addition to localizing the Sl 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.

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.

Stem cell factor is encoded at the Sl 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.

Base pairing involving deoxyinosine: implications for probe design.

The thermal stability of oligodeoxyribonucleotide duplexes containing deoxyinosine (I) residues matched with each of the four normal DNA bases were determined by optical melting techniques. The duplexes containing at least one I were obtained by mixing equimolar amounts of an oligonucleotide of sequence dCA3XA3G with one of sequence dCT3YT3G where X and Y were A, C, G, T, or I. Comparison of optical melting curves yielded relative stabilities for the I-containing standard base pairs in an otherwise identical base-pair sequence. I:C pairs were found to be less stable than A:T pairs in these duplexes. Large neighboring-base effects upon stability were observed. For example, when (X,Y) = (I,A), the duplex is eight-fold more stable than when (X,Y) = (A,I). Independent of sequence effects the order of stabilities is: I:C greater than I:A greater than I:T congruent to I:G. This order differs from that of deoxyguanosine which pairs less strongly with dA; otherwise each deoxyinosine base pair is less stable than its deoxyguanosine counterpart in the same sequence environment. Implications of these results for design of DNA oligonucleotide probes are discussed.

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

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.