Chemical induction of interleukin-8, a proinflammatory chemokine,in human epidermal keratinocyte cultures and its relation to cytogenetic toxicity

Tumor promoters, proinflammatory cytokines, endotoxins, and protein synthesis inhibitors can modulate cell cycle kinetics of various cell types, stimulate production of reactive oxygen species, and induce keratinocytes to produce interleukin-8 (IL-8), a potent chemotactant for polymorphonuclear neutrophils and T lymphocytes. The aim of this study was to determine whether perturbations of cytogenetic responses correlated with the induction of IL-8 expression. Cultures of primary human keratinocytes were grown in serum-free medium with 5 w772/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">mol/L bromodeoxyuridine to label DNA and exposed either to phorbol-13-myristate-12-acetate (PMA) (0.0001–100 ng/ml), cycloheximice (CHX) (0.01–50 w772/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">g), lipopolysaccharide (0.1–100 w772/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">g/ml), tumor necrosis factor-w772/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> (TNFw772/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">) (3.13–50 ng/ml), or interleukin-1w772/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> (IL-1w772/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">) (1–182 pg/ml). Metaphase chromosome preparations were stained by a fluorescence-plus-Giemsa technique to differentiate sister chromatids. For IL-8 production, keratinocytes were grown to 70% confluency and then exposed to chemicals for 24 h. Immunoreactive IL-8 was quantitated from the supernatants by ELISA. With the exception of benzo(a)pyrene used as a positive control, none of the agents induced sister chromatid exchanges. However, PMA and TNFw772/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> induced IL-8 production that coincided with significant cell cycle inhibition. IL-1w772/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> had no effect on cytogenetic endpoints, yet stimulated a 6.3-fold increase in IL-8. CHX inhibited cell cycle progression and mitotic activity at concentrations that were 200 times lower than required for IL-8 induction; however, puromycin (0.31–10 w772/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">g/ml), another protein synthesis inhibitor, did not induce IL-8. At all concentrations tested, TNFw772/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> reduced the mitotic index by w772/xxlarge8764.gif" alt="sim" align="MIDDLE" BORDER="0">45%, slowed cell cycle progression by w772/xxlarge8764.gif" alt="sim" align="MIDDLE" BORDER="0">3.5 h, and induced a flat, albeit large, IL-8 response at concentrations w772/xxlarge8805.gif" alt="ge" align="MIDDLE" BORDER="0">12.5 ng/ml. These agent-specific response patterns suggest that induction of IL-8 production is not always the inevitable result of cell cycle perturbations or genetic damage.Abbreviations B(a)P benzo(a)pyrene - BrdU 5-bromo-2w772/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-deoxyuridine - CHX cycloheximide - ICAM intercellular adhesion molecules - IL-1w772/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> interleukin-1w772/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> - IL-8 interleukin-8 - KGM keratinocyte growth medium - LPS lipopolysaccharide - PKC protein kinase C - PMA phorbol-13-myristate-12-acetate - PMN polymorphonuclear neutrophil - ROS reactive oxygen species - SCE sister chromatid exchange - TNFw772/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> tumor necrosis factor w772/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">     

Skin ultrastructure in relation to prolactin and MSH function in rainbow trout (Oncorhynchus mykiss) exposed to environmental acidification

The present study describes the effects of 14 days exposure to acidified (pH 4.0) soft water in the absence of aluminium, on the ultrastructure of the skin in rainbow trout (Oncorhynchus mykiss). Compared to control fish, there was a moderate increase in the incidence of necrosis in the filament cells of the fish exposed to pH 4.0, but since the integrity of the tissue appeared to be maintained, most of the ultrastructural changes observed may be considered to be adaptive. There was an increase in epidermal thickness, a higher frequency of electrondense vesicles in filament cells, an increase in the undulation of the basal lamina, and the penetration of the epidermis by cytoplasmatic processes of melanocytes in acid-exposed specimens. An infiltration of leucocytes into the epidermis, and the appearance of serous mucous cells, was also evident. Whether these events were under the control of prolactin and/or w5474716/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-MSH, was also investigated, but no indication for activation or inhibition of either prolactin or w5474716/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-MSH producing cells was obtained. Since a previous study (Balm and Pottinger 1993) had demonstrated that plasma cortisol levels were also identical in control and low pH treated trout throughout a 14 day experimental period, it is concluded that under conditions of environmental acidification, the integument autonomically maintains the adjustments necessary for successful acclimation, presumably via paracrine regulatory circuits.     

Interaction of force transmission and sarcomere assembly at the muscle-tendon junctions of carp (Cyprinus carpio): ultrastructure and distribution of titin (connectin) and α-actinin

At muscle-tendon junctions of red and of white axial muscle fibres of carp, new sarcomeres are found adjacent to existing sarcomeres along the bundles of actin filaments that connect the myofibrils with the junctional sarcolemma. As the filament bundles that transmit force to the junction originate proximal to new sarcomeres, they probably relieve these new sarcomeres from premature loading. In red fibres, these filament bundles are long (up to 20 w5v1q23061554/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">m) and dense, permitting light-microscopical immunohistochemistry (double reactions: anti-titin or anti-w5v1q23061554/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-actinin and phalloidin). New sarcomeres have clear I bands; their A band lengths are similar to those of older sarcomeres and the thick filaments lie in register. T tubules are found at the distal side of new sarcomeres but terminal Z lines are absent. The late addition of w5v1q23061554/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-actinin suggests that w5v1q23061554/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-actinin mainly has a stabilizing role in sarcomere formation. The presence of titin in the terminal fibre protrusions is in agreement with its supposed role in sarcomere formation, viz. the integration of thin and thick filaments. The absence of a terminal Z line from sarcomeres with well-registered A bands suggests that this structure is not essential for the anchorage of connective (titin) filaments.     

Glutamine synthetase (GS) expression is reduced in senile dementia of the Alzheimer type

Glutamine synthetase (GS), a metabolic marker of the mature astrocyte, was investigated in the temporal neocortex of postmortem brain samples of 8 cases, either not demented or affected by senile dementia of the Alzheimer type. A negative correlation between the GS protein level and the density of both classical w2m84661/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">A4 deposits and senile plaques was evidenced. Such a correlation for GS underlies a dysfunction of the astroglial metabolism and particularly of the glutamate and ammonia neutralization. Since GS is sensitive to oxidative lesioning, the changes in GS level that were observed, occurring at the posttranslational stage, might reflect oxidative damage and have severe consequences on the pathological cascade of events.     

Interleukin-6 involvement in mesothelioma pathobiology: inhibition by interferon α immunotherapy

A role for interleukin-6 (IL-6) in malignant mesothelioma has been suggested by the clinically presenting symptoms of mesothelioma patients, which include fever, weight loss and thrombocytosis. A murine model of malignant mesothelioma was therefore used to examine the potential role of IL-6 in this cancer type and whether the effect of interferon w03v0/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> (IFNw03v0/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">) therapy on mesothelioma might be mediated, in part, by regulating IL-6 levels and/or IL-6-induced pathobiology. A panel of human and murine mesothelioma cell lines was assayed for endogenous IL-6 production in a bioassay, and for IL-6-mRNA expression. Four out of 5 human and 5 out of 15 murine mesothelioma cell lines produced moderate to high levels of bioactive IL-6 in vitro. This result was corroborated by mRNA detection. One of the representative murine cell lines, AB22, was chosen for further in vivo studies in the murine mesothelioma model. In AB22-inoculated mice detectable serum IL-6 levels were found to precede macroscopically detectable tumour growth, clinical signs (cachexia, abdominal distension, diarrhoea) and changes in the peripheral lymphoid organs (cell depletion and functional depression). Treatment with anti-IL-6 antibody curtailed the clinical symptoms (P<0.001), as did treatment with recombinant human (rhu) IFNw03v0/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> (P<0.001). Neither anti-IL-6 antibody nor rhuIFNw03v0/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> had a direct growth-inhibitory effect on the AB22 mesothelioma cell line in vitro, however, in vivo rhuIFNw03v0/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> treatment of mice inoculated with AB22 cells attenuated both IL-6 mRNA expression in the tumours and serum IL-6 levels, ameliorated the depression of lymphocyte activities, and enhanced the number of tumour-infiltrating lymphocytes and macrophages. On the basis of these results it is suggested that IL-6 mediates some of these effects, directly or indirectly, and that a combination therapy of rhuIFNw03v0/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> and anti-IL-6 antibody may be an improved palliative treatment for patients with malignant mesothelioma.     

Facile removal of the N-indole-mesitylenesulfonyl protecting group using HF cleavage conditions

Summary Nitrogen indole protection of the w8885v/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-methyltryptophan side-chain residue is important for avoiding undesired side reactions during peptide synthesis. Of great importance is the choice of a side-chain protecting group for orthogonal peptide synthesis and its stability under a variety of chemical conditions required for synthesis of the four isomers of this unusual amino acid. We report here the successful use of the mesitylenesulfonyl (Mts) protecting group for w8885v/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-methyltryptophan in the synthesis of melanotropin and CCK peptide analogues and the ready cleavage of this protecting group under HF conditions.     

Somatostatin analogues containing o-aminomethylphenylacetic acid as a bridge unit

Summary A new cis-peptide bond mimetic, w270424416863747/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-benzyl-o-aminomethylphenylacetic acid, was synthesized and incorporated in a homodetic somatostatin analogue. Biological binding tests and 2D NMR conformational analysis indicate that the configuration of the bridge-unit asymmetric center and the orientation of the benzyl side chain play a key role in the biological activity of this type of somatostatin analogues.     

Synthesis and evaluation of azaproline peptides as potential inhibitors of dipeptidyl peptidase IV and prolyl oligopeptidase

Summary A series of azaproline dipeptides with various N-substituents were synthesized as possible active-site-directed inhibitors of two proline-specific serine proteases, dipeptidyl peptidase IV and prolyl oligopeptidase. Compounds with semicarbazide, carbazate, acylhydrazine and sulphonylhydrazine structures were tested. Some compounds show moderate activity, i.e., in the millimolar range.     

A genetic component in human lysosomal enzyme excretion

Acid hydrolases are present in normal human urine in appreciable amounts. Their source appears to be lysosomes released by kidney proximal tubule epithelial cells. For a given lysosomal enzyme the total amount excreted is the product of two parameters, a general one describing the rate of lysosome secretion and a specific one describing the relative concentration of that enzyme in lysosomes. There is considerable population variation in both parameters. Studies of w5jh0632x737v/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase, w5jh0632x737v/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-galactosidase, w5jh0632x737v/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-hexosaminidase, and w5jh0632x737v/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-galactosidase in monozygotic and dizygotic twins show that an appreciable part of this variation is genetic in origin. This appears to be true for both total enzyme excretion and lysosome composition. Although it was not possible to test directly whether this is also true for the rate of lysosome secretion, the fact that the two former parameters are both heritable strongly suggests that the rate of lysosome excretion is also a heritable trait. Taken together with previous data, the results suggest polygenic control of these biochemical traits. It is particularly significant that w5jh0632x737v/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase excretion in normal individuals is a heritable trait since the excretion of this enzyme has frequently been used as a measure of normal and pathological physiological changes.This study was supported by grants from the National Institutes of Health (GM-19521) and the Council for Tobacco Research—U.S.A., Inc. (1080). The work was done while the authors were in the Department of Molecular Biology, Roswell Park Memorial Institute, Buffalo, New York 14263.     

Inheritance of alleles for Cgy1 and Gy4 storage protein genes in soybean

Summary A cultivar lacking the glycinin subunit A₅A₄B₃ (w1w362246435/xxlarge8216.gif" alt="lsquo" align="BASELINE" BORDER="0">Raidenw1w362246435/xxlarge8217.gif" alt="rsquo" align="BASELINE" BORDER="0">) was crossed with one lacking the w1w362246435/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">w1w362246435/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-subunit of w1w362246435/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-conglycinin (w1w362246435/xxlarge8216.gif" alt="lsquo" align="BASELINE" BORDER="0">Keburiw1w362246435/xxlarge8217.gif" alt="rsquo" align="BASELINE" BORDER="0">). Analysis of F₂ and F₃ progeny indicated that the missing bands of the A₅A₄B₃ and the w1w362246435/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">w1w362246435/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-subunit were each controlled by a recessive allele of two independently segregating genes. Gene symbols Gy ₄/gy ₄ and Cgy ₁/cgy ₁ were proposed for the genes which confer the presence or absence of the glycinin and conglycinin subunits, respectively.Cooperative research of USDA-ARS and the Indiana Agric. Exp. Stn., Purdue Univ., West Lafayette, IN 47907, USA. Indiana Agric. Exp. Stn. Journal Article 9675. Financial support from the American Soybean Association Research Foundation is gratefully acknowledged