Kirsten Paludan

1,25(OH)2-D3 is a potent regulator of interleukin-1 induced interleukin-8 expression and production.

Interleukin 8 (IL-8) is a potent leukocyte chemotactic and activating cytokine produced by keratinocytes, fibroblasts, peripheral blood monocytes (PBMC) and endothelial cells. IL-8 is believed to play an important role in the development of inflammation and is thus an obvious target for therapeutical modulation. We studied the possible effect of an endogenous immune modulator 1,25(OH)2-cholecalciferol (1,25(OH)2-D3) on the IL-1-induced IL-8-production by several types of cells. 1,25(OH)2-D3 inhibited the IL-1-alpha induced IL-8 production and mRNA expression in keratinocytes, fibroblasts and PBMC, but not in endothelial cells. Optimal vitamin concentrations varied between 10(-10) and 10(-11) M. These results suggest a potential role of this hormone in the regulation of chemotactic cytokine production.

The chromosomal arrangement of six soybean leghemoglobin genes

Clones containing six leghemoglobin (Lb) genes have been isolated from two genomic libraries of soybean. They encompass two independent DNA regions: a 40‐kb region containing four genes in the order 5′ Lba‐Lbc1‐[unk]Lb‐Lbc3 3′ with the same transcriptional polarity, and another 40‐kb region containing two genes in the order 5′ Lbc4‐Lbc2 3′ with the same polarity. The order in which the Lb genes are arranged in the soybean genome imply that they are activated in the opposite order to which they are arranged on the chromosome. There is a close similarity between corresponding DNA regions outside the Lb genes in the two clusters. Thus, a moderately repetitive DNA element is present in corresponding positions in each cluster. In addition, at least two different non‐Lb genes are linked to each Lb gene cluster in corresponding positions. These genes are apparently regulated in a way which differs from that of the Lb genes. The existence of two very similar Lb gene clusters in soybean suggest that soybean may have evolved from an ancestral form by genome duplication.

Graduated resistance to G418 leads to differential selection of cultured mammalian cells expressing the neo gene.

The effects of Geneticin (G418) selection on the growth and survival of cultured mammalian cells expressing the neomycin-resistance gene (neo) were studied by the analysis of cell clones from two retroviral neo vector-infected populations. We found a correlation between the neo expression level and growth rates in medium containing varying G418 concentrations. This relationship permits the use of differential selection schemes for the isolation of rare cells with increased expression. Comparison, by clone sampling, of vector-positive populations before and after selection with a G418 concentration in the range usually used for selection, showed different expression level and vector copy number distributions for the population infected with the vector of lower LTR activity, but not for the other. Such biasing effects of G418 selection may be important when selected cells are used for quantitative studies of gene expression.

The structure of an unusual leghemoglobin gene from soybean.

A clone containing an unusual leghemoglobin (Lb) gene was isolated from a soybean DNA library present in Charon 4A phage. DNA sequence analysis revealed that the isolated Lb gene has three intervening sequences (IVS‐1, IVS‐2 and IVS‐3) located in the same positions as those found in other Lb genes. Due to a large increase of IVS‐2 and IVS‐3, the isolated Lb gene is about twice the size of a normal Lb gene. The coding sequence derived from the DNA sequence corresponds to no known soybean Lb and attempts to find a corresponding mRNA failed. In addition, the 5′‐flanking sequence of the Lb gene is mutated in two regions which seem to be important for transcription. It is, therefore, tentatively suggested that the isolated Lb gene is non‐functional, and consequently is an Lb pseudogene.

Determination of transient or stable neo expression levels in mammalian cells.

We report an extension of the neomycin phosphotransferase II dot-blot assay to allow more rapid and sensitive quantitative determination of the neo gene product in crude mammalian cell extracts. Our procedure, based upon the dot-blot assay of Platt and Yang [Anal. Biochem. 162 (1987) 502-514], measures both the enzymatic activity and the protein content of a cell extract by scanning with an enzyme-linked immunosorbent assay reader, using the same sample rather than parallel samples for both measurements. We show this assay to be comparable to the chloramphenicol acetyltransferase assay in sensitivity. Therefore, apart from being a useful selectable marker gene, the neo gene is a convenient reporter gene in studies of stable, as well as transient, expression.

Expression of monocyte chemotactic and activating factor (MCAF) in skin related cells. A comparative study

A significant proportion of the infiltrating cells in several inflammatory skin diseases, including psoriasis and allergic contact dermatitis, are monocytes. Additionally, it is known that the cytokine monocyte chemotactic and activating factor (MCAF) can be produced by several cell types present in the skin, suggesting a significant role for MCAF in the accumulation of monocytes during immunological and inflammatory skin reactions. We have recently developed a precise method for quantification of the amount of a specific mRNA species in a given sample and have used this technique to compare specific MCAF mRNA amounts in cultures of human keratinocytes, dermal fibroblasts, endothelial cells, and monocytes, after stimulation with interleukin 1 alpha (IL-1 alpha) for 6 h. Endothelial cells produced very high, monocytes and fibroblasts intermediate, and keratinocytes low amounts of MCAF mRNA. We have also performed time course studies of MCAF mRNA levels in the four cell types. Our findings suggest that the regulation of MCAF mRNA expression in these cells parallels the regulation of the lymphocyte and neutrophil chemotactic factor interleukin 8.

The Soybean Leghemoglobin Genes

Leghemoglobins (Lbs) are myoglobin-like proteins synthesized exclusively in the root nodules which develop through the symbiotic association of Rhizobium with legumes. The function of the Lbs is to facilitate the diffusion of oxygen at a low oxygen tension, thus making the oxygen available for metabolic processes without inactivating the nitrogenase which is very oxygen-sensitive (Bergersen et al., 1973).

ETH615, a synthetic inhibitor of leukotriene biosynthesis and function, also inhibits the production of and biological responses towards interleukin-8

Abstract ETH6I5 (4‐(2‐quinolylmethoxy)‐N‐(3‐fluorobenzyl‐phenyl‐amino‐methyl‐4‐benzoic‐acid), a synthetic inhibitor of leukotriene B4 production and activities, was tested for its effect on the production of and biological responses towards human interleukin‐8. We found that ETH615 inhibits lipopolysaccharide‐induced (LPS‐induced) expression of interleukin‐8 messenger‐RNA (mRNA) and interleukin‐8 production in human peripheral blood mononuclear cells. We also observed that ETH615 completely inhibited interleukin‐8 as well as leukotriene B4 directed chemotaxis of human neutrophils in a dose‐dependent manner. A moderate effect on fMLP‐directed neutrophil chemotaxis was observed. Further, no significant effect on either interleukin‐8. leukotriene B4 or fMLP‐directed T‐cell migration was observed. These results further support the concept of a cytokine‐leukotriene regulatory circuit and encourage the establishment of clinical trials testing the effect of ETH615 on inflammatory skin diseases, which are characterized by high levels of interleukin‐8 and leukotriene B4 in lesional skin.

Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay

Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene neo; hph and neo may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.