Spermine down-regulates superoxide generation induced by fMet-Leu-Phe in electropermeabilized human neutrophils.

Effect of spermine, a naturally occurring polyamine, was investigated on superoxide generation in intact and electropermeabilized human neutrophils. Spermine suppressed N-formyl-methionyl leucyl phenylalanine (fMLP)-induced superoxide generation in permeabilized cells by reducing the rate and shortening the duration time. The inhibition was specific for spermine comparing with its precursor amines, spermidine and putrescine. The inhibition was not observed when cells were preincubated with spermine without permeabilization. Concanavalin A-induced superoxide generation was also down-regulated by spermine in permeabilized cells, but the activation induced by non receptor-mediated agonist (dioctanoylglycerol, phorbol myristate acetate, and arachidonate) was not affected by spermine. On the other hand, GTP-gamma-S-induced activation of superoxide generation was substantially suppressed by spermine. These results indicate that spermine inhibition occurs at a step prior to protein kinase C in signal transduction or in a pathway which is independent of the kinase.     

Spermine cytotoxicity to human colon carcinoma-derived cells (CaCo-2)

Spermine is a constituent of all vertebrate cells. Nevertheless, it exerts toxic effects if it accumulates in cells. Spermine is a natural substrate of the FAD-dependent polyamine oxidase, a constitutive enzyme of many cell types. It has been reported that the toxicity of spermine was enhanced if polyamine oxidase was inhibited. We were interested to examine spermine toxicity to human colon carcinoma-derived CaCo-2 cells because, in contrast to most tumor cell lines, CaCo-2 cells undergo differentiation, which is paralleled by changes in polyamine metabolism. CaCo-2 cells were remarkably resistant to spermine accumulation, presumably because spermine is degraded by polyamine oxidase at a rate sufficient to provide spermidine for the maintenance of growth. Inactivation of polyamine oxidase increased the sensitivity to spermine. A major reason for the enhanced spermine cytotoxicity at low polyamine oxidase activity is presumably the profound depletion of spermidine, and the consequent occupation of spermidine binding sites by spermine. Hydrogen peroxide and the aldehydes 3-aminopropanal and 3-acetamidopropanal, the products of polyamine oxidase-catalyzed splitting of spermine and N ¹-acetylspermine, contribute little to spermine cytotoxicity. Activation of caspase by spermine was insignificant, and the formation of DNA ladders, another indicator of apoptotic cell death, could not be observed. Thus it appears that cell death due to excessive accumulation of spermine in CaCo-2 cells was mainly nonapoptotic. The content of brush border membranes did not change between days 6 and 8 after seeding, and it was not affected by exposure of the cells to spermine. However, the activities of alkaline phosphatase, sucrase, and aminopeptidase in nontreated cells were considerably enhanced during this period, but remained low if cells were exposed to spermine. These changes appear to indicate that differentiation is prevented by intoxication with spermine, although other explanations cannot be excluded. This revised version was published online in July 2006 with corrections to the Cover Date.     

Endothelin-converting enzyme in human tissues

Our aim was to determine whether the expression of endothelin-converting enzyme in human tissues would correlate with the distribution of its substrate, big endothelin-1, and its product, the mature peptide. Site-directed antisera raised against the conserved C-terminus of the mammalian enzyme were used to measure the immunoreactive enzyme in microsomal fractions prepared from tissue homogenates and to localize staining to the endothelial cells lining large conduit and smaller resistance vessels within cardiac, adrenal, respiratory and brain tissue. The activity of endothelin-converting enzyme was measured and characterized in isolated endothelial cells. This pattern of staining in the vascular endothelium paralleled that of mature endothelin and big endothelin-1, and these peptides were detectable by radioimmunoassay in all tissues examined. Immunoreactive endothelin-converting enzyme localized to other cell types, including bronchial epithelial cells, and to fibres within the glial limitans, neuronal processes and cell bodies of the cerebral cortex. Although perivascular astrocytes in the subcortical white matter displayed intense endothelin-converting enzyme-like immunoreactivity, endothelin staining was not detected. The results suggest that endothelin-converting enzyme has a ubiquitous distribution within the human vascular endothelium and is positioned to catalyse the conversion of big endothelin-1 to the biologically active endothelin-1, which on release may contribute to the maintenance of basal tone in humans. Endothelin-converting enzyme localized to epithelial cells in peripheral tissues or astrocytes within the brain may be upregulated in pathophysiological conditions in which endothelin levels are increased and could represent a further target for therapeutic intervention by enzyme inhibitors. © 1998 Chapman & Hall     

Selenoproteins in human thyroid tissues

The aim of the present work was to clarify whether the activities of selenoenzymes can serve as markers for different tumors or goiters, as classified by histological criteria. The following parameters were determined: 1) selenium content of plasma (Se), 2) activities of the selenoenzymes: plasma glutathione peroxidase (plGSHPx), cytosolic glutathione peroxidase (cGSHPx), type I and type II iodothyronine deiodinases (ID-I, ID-II), thioredoxin reductase (THRR) in human thyroid tissues. The material came from follicular neoplasm, papillary carcinoma, struma nodosa, struma lymphomatosis Hashimoto, other thyroid surgery specimens, and normal tissues. There was no difference in Se nor in plGSHPx between patients and healthy volunteers. No significant differences were found for any parameter in thyroid carcinoma versus normal or goitrous thyroid tissue. In the whole group of thyroid surgery specimens the statistically significant correlations were found between ID-I and ID-II and between THRR and selenoperoxidases. Principal components analysis confirmed the above correlation and moreover revealed correlation between Se and plGSHPx, but did not detect any clear distinction between patients with the different diagnoses.     

Spermine, a natural polyamine, suppresses LFA-1 expression on human lymphocyte

Natural polyamines, spermine, spermidine, and putrescine, play a pivotal role in the regulation of gene expression; therefore, the age-dependent decreases and the disease-dependent increases in polyamine synthesis suggest a possible contribution of polyamines to the age-related and disease-associated changes in cellular function. In this study, we examined the effects of polyamines on the cellular function and the expression of adhesion molecules on human PBMCs from healthy volunteers. Flow cytometry revealed that PBMCs cultured with spermine decreased mean fluorescent intensities (MFIs) of CD11a and CD18 in the lymphocyte light-scattered region, but not in the monocyte region. This suppression was observed in a dose- and time-dependent manner and found nonspecifically on all cell subsets we tested (CD3(+), CD4(+), CD8(+), CD19(+), CD45RA(+), CD45RO(+), CD4(+)CD45RA(+), CD4(+)CD45RO(+), CD8(+)CD45RA(+), CD8(+)CD45RO(+)). The decreases of CD11a and CD18 MFIs were accompanied by the decrease in adherent capacity of PBMCs to HUVECs. Spermine did not hinder cell activities or cell viability. Among 42 healthy volunteers (mean, 49.5 years old; from 26 to 69), blood spermine levels inversely correlated with the CD11a MFIs of cells in the lymphocyte region (r = -0.48; p = 0.001), but not with those in the monocyte region. The effects of spermidine seemed weaker than those of spermine, and blood spermidine levels had no correlation with CD11a MFIs of the lymphocyte region. Putrescine had no effect on the expressions of membrane molecules. Polyamines, especially spermine, decrease LFA-1 (CD11a/CD18) expression on human lymphocyte and adhesion capacity of PBMCs to HUVECs.     

Somatic mosaicism in healthy human tissues

From the fertilization of an egg until the death of an individual, somatic cells can accumulate genetic changes, such that cells from different tissues or even within the same tissue differ genetically. The presence of multiple cell clones with distinct genotypes in the same individual is referred to as 'somatic mosaicism'. Many endogenous factors such as mobile elements, DNA polymerase slippage, DNA double-strand break, inefficient DNA repair, unbalanced chromosomal segregation and some exogenous factors such as nicotine and UV exposure can contribute to the generation of somatic mutations, thereby leading to somatic mosaicism. Such changes can potentially affect the epigenetic patterns and levels of gene expression, and ultimately the phenotypes of cells. Although recent studies suggest that somatic mosaicism is widespread during normal development and aging, its implications for heightened disease risks are incompletely understood. Here, I discuss the origins, prevalence and implications of somatic mosaicism in healthy human tissues.     

Tetranectin immunoreactivity in normal human tissues

Summary A recently discovered human plasma protein, tetrancetin (TN), which has previously been demonstrated immunohistochemically within various endocrine tissues, was in this study identified in an additional number of epithelial and mesenchymal cells by two polyclonal antibodies and one monoclonal using the conventional immunoperoxidase staining technique and a modification of the CLONO-GLAD procedure. TN was found in endothelial and epithelial tissues, particularly in cells with a high turn-over or storage function such as gastric parietal and zymogenic cells absorptive surface epithelium of the small intestine, ducts of exocrine glands and pseudostratified respiratory epithelium. Also mesenchymal cells produced a TN positive staining reaction, which was most conspicious in mast cells, but also present in some lymphocytes, plasma cells, macrophages, granulocytes, striated and smooth muscle cells and fibroblasts. SDS-PAGE and Western blotting analysis of cultured human embryonal fibroblasts (WI-38) showed that the cells besides TN contain another protein with a molecular weight of 82 000. As this protein, however, reacted with our affinity purified antibodies it probably represents a precussor of TN or a protein with a molecular weight of approximately 60 000, which is covalently linked to TN. This and the fact that TN shows amino acid sequence homologiess to the carboxyterminal part of the asialo-glycoprotein receptor and a cartilage proteoglycan core protein as well a binding affinity to plasminogen points to TN as being part of a larger molecule, which possibly has been cleaved by proteolysis at the cellular site and then passed into the blood, where it polymerizes into a tetramer.     

AMP deaminase isozymes in human tissues

In human, there are four AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) isozymes: E1, E2, M and L. Chromatographic, electrophoretic and immunological studies showed the existence of isozymes E1 and E2 in erythrocytes, isozyme M in muscle and isozyme L in liver and brain. The tissues such as heart, kidney and spleen contained isozymes E1, E2 and L. Isozymes E1, M and L were isolated as apparently homogeneous preparations. The three isozymes were all tetramers composed of identical subunits, but differing slightly in molecular weight; isozyme E1 showed a subunit molecular weight of 80 000, isozyme M 72 000 and isozyme L 68 000. They were immunologically different from one another. The antisera precipitated only the corresponding enzyme and did not precipitate any other isozyme. The three isozymes were also different in kinetic and regulatory properties. Isozyme E2 was very similar to isozyme E1 in immunological and kinetic properties, although isozyme E2 could be separated from isozyme E1 by phosphocellulose chromatography, and zonal electrophoresis.     

Lysine-ketoglutarate reductase in human tissues.

Lysine-ketoglutarate reductase (saccharopine dehydrogenase (NADP+, lysine-forming) EC 1.5.1.8) from human liver has been partially purified and characterized. A spectrophotometric assay is described. The Michaelis constants have been determined for lysine (1.5-10-3 M), alpha-ketoglutarate (1-10-3 M) and NADPH (8-10-5 M). The pH optimum is 7.8. The enzyme is product inhibited. The specificity of the enzyme, response to inhibitors, pH and thermal stability are reported. Lysine-ketoglutarate reductase is present in high concentration in liver and heart, to a lesser degree in kidney and skin and in trace amounts in several other tissues. Saccharopine dehydrogenase (saccharopine dehydrogenase (NAD+, L-glutamate-forming) EC 1.5.1.9) was demonstrable only in liver and kidney. Lysine-ketoglutarate reductase reacts effectively with delta-hydroxylysine.     

Spermine accumulation under salt stress

Polyamines have long been recognized to be linked to stress situations, and it is generally accepted that they have protective characteristics. However, little is known about their physiological relevance in plants subjected to long-term salt stress. In order to precise their importance, two rice (Oryza sativa) cultivars differing in their salt tolerance were salinized for 7, 14 and 21 days. The activities of some of the enzymes involved in polyamine metabolism, free polyamines and proline contents were evaluated. Arginine decarboxylase and S-adenosyl-L-methionine decarboxylase activities were reduced in both cultivars as a consequence of salt treatment. However, spermidine synthase activity was reduced in the salt tolerant cultivar (var Giza) but not in the salt sensitive (var El Paso), while no polyamine oxidase activity was detected. During the salinization period, putrescine and spermidine levels decreased in both cultivars, although less dramatically in Giza. Simultaneously, spermine accumulations occur in both varieties, while proline accumulation was major in the sensitive one. However, spermine accumulation induced by treatment with spermidine synthase inhibitor cyclohexylamine, determined no reduction in leaf injury associated with salt stress in both cultivars. The data presented suggest that spermine accumulation is not a salt tolerance trait.