Effects of platelet-derived growth factor and fibroblast growth factor on free intracellular calcium and mitogenesis

Although increased free intracellular calcium (Cai) may be one of the main regulators of cell growth and differentiation, studies in cell populations have implied that not all growth factors produce Cai increases. In order to examine in more detail whether Cai increases were related to mitogenesis, we used digital image analysis of intracellular Fura-2 fluorescence to measure Cai in individual BALB/c 3T3 cells stimulated with either platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF). We found that PDGF induced larger and more prolonged Cai increases than FGF did, but that both growth factors induced an initial rapid increase in Cai (less than 2 min) followed by a later sustained increase (greater than 20 min). Only the prolonged Cai increase required extracellular calcium. Following PDGF treatment (1-8 units/ml), the percentage of cells with a large peak Cai increase (greater than twofold) correlated with the percentage of cells made competent (subsequent growth in 1% platelet-poor-plasma). In contrast, purified bovine basic FGF (200-800 pg/ml) and recombinant human acidic FGF (10-300 ng/ml) produced peak Cai increases that were not directly correlated with mitogenesis. In addition, concentrations of intracellular Quin 2 that inhibited Cai transients also inhibited PDGF stimulation but not FGF stimulation of mitogenesis. Thus, Cai increases are necessary for mitogenesis in BALB/c 3T3 cells stimulated by PDGF, but not that stimulated by FGF.     

Identification of tyrosine-phosphorylated colony-stimulating factor 1 (CSF-1) receptor and a 56-kilodalton protein phosphorylated in intact human cells in response to CSF-1

Colony-stimulating factor 1 (CSF-1) selectively supports the survival, proliferation, and maturation of hemopoietic cells of the monocyte/macrophage lineage. Although the cellular receptor for CSF-1, (the c-fms protein) is a protein-tyrosine kinase activated by the binding of CFS-1, the role of phosphorylation of cellular proteins in CSF-1 signal transduction is poorly understood. Therefore, we examined the CSF-1-stimulated phosphorylation of cellular proteins in human BeWo choriocarcinoma cell line (known to express the c-fms protein). BeWo cells were metabolically labeled with 32Pi, stimulated with recombinant human CSF-1, and extracted with detergent. Phosphotyrosyl proteins were isolated from detergent extracts by affinity chromatography on a highly specific antibody to phosphotyrosine. Rapid phosphorylation of 170-kd protein, followed closely by the phosphorylation of a 56-kd protein, was observed in response to CSF-1. The 170-kd phosphotyrosyl protein bound to wheat germ agglutinin and was secondarily immunoprecipitated with a specific anti-fms serum, consistent with its identity as the CSF-1 receptor. Although purified human macrophages that proliferate in culture in response to CSF-1 are not generally accessible, CSF-1 did stimulate the phosphorylation of a 56-kd protein in intact mononuclear leukocytes from human peripheral blood. Thus, the BeWo cell line may represent a good model for the study of CSF-1-stimulated cellular protein phosphorylation.     

Heparin-binding growth factors and their receptors

Heparin-binding growth factors modulate diverse biological activities including cellular proliferation, cellular differentiation, morphogenesis, and angiogenesis. Biochemical characterization for two members of the heparin-binding growth factor family, acidic and basic fibroblast growth factors, is extensive, while characterization of the remaining five members is forthcoming. Cell surface receptors have been identified for acidic and basic fibroblast growth factors, but little is known concerning their sites of action in vivo or the mechanisms involved in transducing the energy of growth factor binding to a biological response. An understanding of the biological basis for the diversity of the heparin binding growth factor family and the in vivo actions of these factors will prove a major challenge to future research efforts.     

Opsonizing properties of natural antibodies of rainbow trout, Oncorhynchus mykiss (Walbaum)

In order to confirm previous observations in which a protective effect of rainbow trout natural antibodies against furunculosis was suspected, phagocytosis studies wereconducted in vitro , using combinations of rainbow trout sera with high or low levels of natural antibodies and active or inactivated complement as opsonizing factors. Opsonization was observed in all the cases where complement was present, and to a lesser degree with sera containing only natural antibodies. The results confirm the prime importance of the complement system and provide additional evidence for a possible role of natural antibodies in antimicrobial defences.     

Studies on the Effect of Insulin-Like Growth Factor-I on Catecholamine Secretion from Chromaffin Cells

Chromaffin cells cultured in serum-free medium secreted a smaller percentage of their catecholamine stores in response to stimulation by high K+ (55 mM) than did cells cultured in serum-containing medium. Addition of insulin-like growth factor-I (IGF-I) to serum-free medium restored high K(+)-stimulated catecholamine secretion to the levels seen in serum-treated cultures. In contrast, addition of IGF-I to serum-containing medium had little effect on catecholamine secretion. These results suggest that serum contains IGF-I or another factor that maintains the secretory responsiveness of chromaffin cells. IGF-I not only enhanced high K(+)-stimulated catecholamine secretion, but also augmented secretion elicited by the nicotinic agonist dimethyl-phenylpiperazinium, the dihydropyridine agonist Bay K 8644, and Ba2+. IGF-I did not affect the dependence of catecholamine secretion on extracellular Ca2+ concentration nor did it affect the time course of secretion. Experiments using 45Ca2+ demonstrated that IGF-I treatment enhanced Ca2+ uptake into the cells. When cells were permeabilized by treatment with digitonin, Ca2(+)-dependent catecholamine secretion was slightly, but consistently, greater from IGF-I-treated cells than from untreated cells. Our results suggest that IGF-I may enhance catecholamine secretion partly by increasing Ca2+ entry into the cells and partly by affecting a step distal to Ca2+ entry.     

A comparison of within-plant karyological heterogeneity between agamospermous and sexualTaraxacum (Compositae) as assessed by the nucleolar organiser chromosome

Morphological variation for the NOR chromosome was studied for four half-siblings of a sexual outbreedingTaraxacum, for three siblings of the obligate agamospermT. pseudohamatum, and for two individuals of the agamospermT. brachyglossum. No rearrangement was detected for the 113 chromosomes of sexuals, or for 41 chromosomes of two agamospermous individuals. In the other three agamospermous individuals, 3/16, 5/50, and 5/20 chromosomes showed evidence of chromosomal rearrangement. The majority of rearrangement events (10/13) occurred to the satellite rather than to the body of the NOR-chromosome. It is considered that such high levels of somatic chromosomal rearrangement in agamospermousTaraxacum may be the result of activity by transposable genetic elements. This recombination may be of selective advantage to asexual plants which cannot generate genetic variability through the sexual process.     

Genetic variation and population structure of cod, Gadus morhua L., in some fjords in northern Norway

Biochemical genetic polymorphism in cod from several fjords in Troms, northern Norway was analysed. Gene frequencies at several polymorphic loci ( Hb, Pgi, Ldh, Pgm, Gpd and Idh ) are given. Significant variation was found both within and between fjords, and even between samples from the same locality sampled in different years, indicating a mosaic structure of the cod population in the area studied. The results of the haemoglobin variation are compared to similar results obtained more than 20 years ago in the same and adjacent areas. The biochemical genetic variation is discussed in relation to stability of gene frequencies, isolation mechanisms and migration patterns.     

Partial characterization of the trait for enhanced K+−Na+ discrimination in the D genome of wheat

The long arm of chromosome 4D of wheat (Triticum aestivum L.) contains a gene (or genes) which influences the ability of wheat plants to discriminate between Na⁺ and K⁺. This discrimination most obviously affects transport from the roots to the shoots, in which less Na⁺ and more K⁺ accumulate in those plants which contain the long arm of chromosome 4D. Concentrations of Na⁺ and K⁺ in the roots, and Cl⁻ concentrations in the roots and shoots, are not significantly affected by this trait, but Na⁺, K⁺ and Cl⁻ contents of the grain are reduced. The trait operates over a wide range of salinities and appears to be constitutive. At the moment it is not possible to determine accurately the effect of this trait on growth or grain yield because the aneuploid lines which are available are much less vigorous and less fertile than their euploid parents.     

Transformed plants with elevated levels of chloroplastic SOD are not more resistant to superoxide toxicity

The petunia nuclear gene which encodes the chloroplast isozyme of superoxide dismutase, SOD-1, has been fused with an efficient rbcS promoter fragment and 3 flanking region and introduced into tobacco and tomato cells. Transformed plants carrying this chimeric gene have up to 50-fold the levels of SOD-1 which occur in wild-type plants. However, tobacco plants with 30-to 50-fold the normal SOD-1 activity do not exhibit resistance to the light-activated herbicide paraquat. Similarly, tomato plants with 2-to 4-fold increases in SOD-1 do not exhibit tolerance to photoinhibitory conditions known to increase superoxide levels (high light, low temperatures and low CO₂ concentrations). Our data indicate that increasing the chloroplastic SOD level in a plant cell is not sufficient to reduce the toxicity of superoxide.     

Restriction Enzyme Analysis of Mitochondrial DNA of Acanthamoeba Strains in Japan

ABSTRACT. Eight isolates, identified as either Acanthamoeba castellanii or A. polyphaga from human eye infections, contact lens containers, and soil in Japan, were characterized by restriction fragment length polymorphisms (RFLP) of mitochondrial DNA (mtDNA). Mitochondrial DNA was digested with either Bgl II, Eco R I, Hind III, Hpa I, Sca I or Xba I, electrophoresed in agarose gels, and stained with ethidium bromide. Four distinct RFLP phenotypes that refer to the collection of six fragment size patterns obtained for a single strain with six enzymes, were discovered among the eight strains used in this study. Three strains morphologically classified as A. polyphaga share a single RFLP phenotype with the Ma strain of A. castellanii. The interspecific sequence differences of 7.06–12.74% in DNA nucleotide were estimated from the proportion of DNA fragments shared by each pair of mtDNA.