Induction of the murine “W phenotype” in long-term cultures of human cord blood cells by c-kit antisense oligomers

The murine white (W) spotting locus is the site of the c-kit gene and encodes a tyrosine kinase receptor while the complementary Steel (Sl) iocus encodes its ligand. Mutations at either locus have profound effects on hematopoiesis, particularly erythroid and mast cell proliferation. We added c-kit antisense oligonucleotides to long-term suspension cultures of enriched human umbilical cord progenitor cells. This resulted in the suppression of c-kit gene expression and the preferential suppression of the generation of erythroid burst-forming cells (BFU-E) which extended over the life of the culture (3 weeks). The results provide an in vitro model of the “W phenotype” in human hematopoiesis and confirm the importance of c-kit gene function in early erythropoiesis. Because the generation of BFU-E was suppressed even after c-kit gene expression had recovered, this gene product may be critical to the erythroid commitment process. © 1993 Wiley-Liss, Inc.     

Cellular Quantification of Tyrosine Hydroxylase in the Rat Brain by Immunoautoradiography

Abstract: We developed a rapid and sensitive radioimmunohistochemical method for the quantification of tyrosine hydroxylase (TH) at both the anatomical and cellular level. Coronal tissue sections from fresh-frozen rat brains were incubated in the presence of a TH monoclonal antibody. The reaction was revealed with a ³⁵S-labeled secondary antibody. TH content was quantified in catecholaminergic brain areas by measuring optical density on autoradiographic films or silver grain density on autoradiographic emulsion-coated sections. Regional TH concentrations determined in the locus ceruleus (LC), substantia nigra pars compacta (SNC), and ventral tegmental area (VTA) were significantly increased by 45% after reserpine treatment in the LC but unchanged in the SNC and VTA. Microscopic examination of TH radioimmunolabeling showed a heavy accumulation of silver grains over catecholaminergic cell bodies. In the LC, grain density per cell was heterogeneous and higher in the ventral than in the dorsal part of the structure. After reserpine treatment, TH levels were significantly increased (57%) in the neurons of the LC but not in those of the SNC or VTA. The data support the validity of this radioimmunohistochemical method as a tool for quantifying TH protein at the cellular level and they confirm that TH protein content is differentially regulated in noradrenergic and dopaminergic neurons in response to reserpine.     

Characterization of hemocytes from the marine amphipod Parhyale hawaiensis (Dana 1853): Setting the basis for immunotoxicological studies

Hemocytes are circulating blood cells that play a crucial function in amphipods and other crustacean immune systems. The hemocytes of the marine tropical amphipod Parhyale hawaiensis have been used for the evaluation of DNA damage and micronuclei, but they have not been characterized in the scientific literature. The aim of this study was to describe the hemolymph cells of P. hawaiensis and study their phagocytotic activity. Basic dyes were used to differentiate the cell types and the presence of lipids. The total hemocyte counts (THCs) and the proportion and sizes of the hemocyte types were determined. Hemolymph was exposed to Escherichia coli for verification of the presence of phagocytosis. Three cell types, all containing lipids, were identified in P. hawaiensis: granulocytes (oval shape, 13.4 × 7.6 μm), semi-granulocytes (oval shape, 14.1 × 7.2 μm), and hyalinocytes (round shape, 9.6 × 7.2 μm). Those three cell types were found in different percentages in males (64.8%, 31.1%, and 4.2%) and females (70.1%, 28.2%, and 1.7%). THCs for males were 9007 ± 3800 cells per individual and 4695 ± 1892 cells per individual for females. The cells of E. coli were phagocytized by the hemocytes. Our findings increased the knowledge of hemocytes in P. hawaiensis and is a step forward in using hemocyte-based immune responses as an endpoint in ecotoxicology.     

Nickel-induced increases in gap junctional communication in the uterine cell line SK-UT-1

Summary Previous studies have suggested that gap junctions may have a role in various uterine functions, including parturition. Because nickel has been demonstrated to increase uterine contractility in vitro, the effect of nickel (II) chloride on gap junctional communication was assessed in a tumorigenic uterine cell line, SK-UT-1 (ATCC HTB 114). Cells were exposed in vitro to 25 and 50 μM NiCl₂ for 24 h or 100 μM NiCl₂ for 3, 12, and 24 h, then functional gap junctional communication was measured as the transfer of Lucifer yellow dye from microinjected donor cells to their primary neighbor cells. Dye transfer was significantly increased only in cell cultures exposed to 100 μM NiCl₂ for 24h, compared to untreated controls, lower doses, and shorter exposure periods. This response was inhibited by the simultaneous co-treatment of SK-UT-1 cells with magnesium by adding 100 μM MgSO₄ to the dosing medium. Possible mechanisms and implications for these findings are discussed.     

Cold-induced Chromosome Regions and Karyosystematics in Sambucus and Viburnum

The genera Viburnum, Sambucus and Lonicera have been investigated for chromosome number and karyomorphology including Giemsa-C-banding, fluorochrome (DAPI/CMA) banding and cold treatment. Cold-induced undercontracted chromosome regions (CIRs) are found in Viburnum and Sambucus for the first time and are apparently identical with larger hc regions, shown by Giemsa C-banding. Certain narrow C-bands are not cold-sensitive. CIRs frequently react brightly CMA-positive in Viburnum and Sambucus, while DAPI fluorescence is virtually ineffective. The occurrence of CIRs within plants is possibly linked to certain nuclear characters such as large chromosomes and continuous condensation behaviour. Cold-induction has possibly also some influence on euchromatin condensation characteristics in prophasic chromosomes. Several karyological characters point to a closer relationship between Viburnum, Sambucus and Adoxa: Relatively large chromosomes, continuous condensation behaviour, reticulate to semireticulate interphase nuclei and presence of CIRs. These genera appear isolated from Lonicera and the Caprifoliaceae s.str., which differ remarkably in karyomorphology.     

Regulated expression of the rat medium chain hydrolase gene in transgenic rape seed

Medium chain hydrolase (MCH) is an enzyme which regulates the chain length of fatty acid synthesis specifically in the mammary gland of the rat. During lactation, MCH interacts with fatty acid synthase (FAS) to cause premature release of acyl chains, thus providing medium chain fatty acids for synthesis of milk fat. In this study we have investigated the ability of rat MCH to interact with the phylogenetically more distant FAS structure present in plant systems and to cause a perturbation of fatty acid synthesis. Inin vitro experiments, addition of purified MCH to rapeseed homogenates was found to cause a significant perturbation of fatty acid synthesis towards medium chain length products. The rat MCH gene was expressed in transgenic oilseed rape using a seed specific rape acyl carrier protein (ACP) promoter and a rape ACP plastid targeting sequence. Western analysis showed MCH protein to be present in transgenic seed and for its expression to be developmentally regulated in concert with storage lipid synthesis. The chimaeric preprotein was correctly processed and immunogold labelling studies confirmed MCH to be localized within plastid organelles. However, fatty acid analysis of oil from MCH-expressing rape seed showed no significant differences to that from control seed.