Synergistic interaction of a cytokine produced by embryonic fibroblasts and a lymphokine contained in Con A-stimulated spleen cell culture for murine macrophage differentiation: a model experiment using cell line cells, M1

The modulating activity of the culture supernatant of Con A-stimulated murine spleen cells for macrophages was investigated by using M-1 cells, which could differentiate into macrophage-like cells (referred to as M+1 hereafter), cocultured in a conditioned medium (CM) containing a differentiation factor (DF) obtained from the secondary culture of murine embryonic fibroblasts. DF induced Ia antigens on M-1 cells at a high rate in parallel with the appearance of Fc-receptor (FcR)-dependent phagocytic activity for erythrocytes coated with an antibody (EA). In contrast, Con A-sup alone had no modulating effect on M-1 cells. However, the Con A-sup stimulates synergistically M-1 cells with DF. Thus, coculture of M-1 cells with Con A-sup and DF generates M++1 cells which possess higher phagocytic activity than M+1 cells. These cells also exhibited stronger accessory cell activity than M+1 cells when tested for their promoting effect on IL-2 production by Sephadex G-10-passed spleen cells. The accessory cell activity of M++1 cells was further enhanced by culture with lymphocytes in the presence of indomethacin while that of M+1 cells did not change. These findings suggest that M-1 cells probably acquire potentiating, as well as inhibitory activity at the same time when cultured with DF and Con A-sup. The functional maturation caused by Con A-sup seemed to be associated with the expression of a receptor for a lymphokine, termed phagocytosis-augmenting factor (PAF) which is present in the Con A-sup. Such a receptor appeared to be common to macrophage lineage, since PAF in Con A-sup was absorbed out with splenic adherent cells and peritoneal exudate cells (PEC) in addition to M+1 cells, but not with nonadherent splenic lymphocytes or lymphoid cell line cells, such as EL-4 and L-1210. This fact suggests that PAF is different from interferon-gamma (IFN) which is known to modulate the function of lymphocytes. Inability of PAF to bind Cibacron Blue-Sepharose, unlike IFN, supports this notion. The molecular weight of PAF is approximately 2-3 X 10(4). Thus, the present studies suggest the requirement of at least two signals for the full maturation of macrophages, a cytokine represented by DF and a lymphokine, by PAF.     

Methyl cinnamate derivatives enhance UV-induced mutagenesis due to the inhibition of DNA excision repair in Escherichia coli B/r

UV-induced mutagenesis in Escherichia coli B/r WP2 was enhanced by certain derivatives of methyl cinnamate which themselves were not mutagenic. Methyl ferulate, methyl isoferulate and methyl sinapate showed this effect markedly. Such an enhancement effect was absent with the derivatives of cinnamic acid and ethyl cinnamate and was not observed in Escherichia coli WP2s uvrA. Methyl sinapate also enhanced 4NQO-induced mutation and suppressed liquid-holding recovery in the above repair-proficient strain. The presence of methyl sinapate in plating agar medium decreased the survival of UV-irradiated cells of a recombination-repair-deficient strain, CM571 recA. However, the effect was not observed with those of WP2s uvrA. In an in vitro experiment in which the removal rate of thymine dimers was measured, methyl sinapate clearly inhibited this repair event. From these results, we conclude that methyl sinapate inhibits DNA excision repair, thus enhancing UV mutagenicity.     

Isolation and characterization of a third proteoglycan (PG-Lt) from chick embryo cartilage which contains disulfide-bonded collagenous polypeptide

Chick embryo epiphyseal cartilage has been shown to contain three different proteoglycan species (PG-H, PG-Lb, and PG-Lt). This report is concerned with the purification and characterization of the third proteoglycan, PG-Lt. The proteoglycan can be separated from the other two by virtue of its low buoyant density in a CsCl density gradient and further purified by consecutive ion exchange and gel chromatography. The final preparation is composed of PG-Lt monomer and PG-Lt oligomer. The amino acid composition of PG-Lt is quite different from that of PG-H and PG-Lb and rather resembles that of collagens with respect to high content of glycine and high degrees of hydroxylation of proline and lysine. PG-Lt monomer is composed of disulfide-bonded subunits of Mr congruent to 120,000 and 190,000 as demonstrated by its gel electrophoretic behavior after reduction with 2-mercaptoethanol. The latter, but not the former, contains dermatan sulfate chains with glucuronic acid/iduronic acid residues and yields a protein-enriched core molecule of Mr congruent to 100,000 after digestion with chondroitinase ABC. Both of the protein subunits are completely digestible with bacterial collagenase. Immunofluorescence microscopic examination of cartilage tissues, using an antibody against PG-Lt, shows that this proteoglycan exists in both the cartilage matrix and perichondrial noncartilagenous region. When chondrocytes are plated onto tissue culture dishes, the antibody stains strands found on the cell surfaces and in the intercellular space of substrate-attached cell layers, suggesting that PG-Lt mediates cell-to-cell and cell-to-substrate contacts.     

蚕豆叶片发育与衰老过程中超氧物歧化酶活性与丙二醛含量变化

蚕豆植株叶片随茎节自上而下表现出明显的发育与衰老顺序,可作为衰老特征的是叶绿素和蛋白质含量明显下降。蚕豆叶中SOD活性主要定位于12 000× g离心后所得的上清液和叶绿体组分。衰老叶片的SOD总活性和叶绿体组分的相对活性都有所下降,SOD同工酶谱也发生了改变。O_2~ 产生速率随叶龄增大而稍上升;而MDA含量在叶片外观表现枯黄衰老征兆前就急剧上升。可能因为衰老叶片过氧化氢酶活性大幅度下降与SOD之间的不平衡,致使O_2~ 代谢中间产物累积而引起膜的损伤.     

Physical effects of negative air ions in a wet sauna

 The physical effects of negative air ions on humans were determined in an experimental sauna room equipped with an ionizer. Thirteen healthy persons took a wet sauna bath (dry bulb temperature 42° C, relative humidity 100%, 10 min exposure) with or without negative air ions. The subjects were not told when they were being exposed to negative air ions. There were no differences in the moods of these persons or changes in their blood pressures between the two saunas. The surface temperatures of the foreheads, hands, and legs in the sauna with negative ions were significantly higher than those in the sauna without ions. The pulse rates and sweat produced in the sauna with ions were singificantly higher than those in the sauna without ions. The results suggest that negative ions may amplify the effects on humans of the sauna. Received: 31 March 1995 / Revised: 25 July 1995 / Accepted: 26 July 1996     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.      

Noisy-threshold control of cell death

Background  

Cellular responses to death-promoting stimuli typically proceed through a differentiated multistage process, involving a lag phase, extensive death, and potential adaptation. Deregulation of this chain of events is at the root of many diseases. Improper adaptation is particularly important because it allows cell sub-populations to survive even in the continuous presence of death conditions, which results, among others, in the eventual failure of many targeted anticancer therapies.     

A robust, target-driven, cell-based assay for checkpoint kinase 1 inhibitors

Checkpoint kinase 1 (Chk1), a serine/threonine kinase, plays an important role in DNA damage checkpoint control and is an attractive target for cancer treatment. To develop a Chk1-specific cell-based assay, stable clones were established in which Chk1 kinase domain fused at its N-terminus with p53 through 4 tandem repeats of Gly-Gly-Gly-Gly-Ser was expressed in an inducible manner. Chk1 kinase specificity of the phosphorylation of fused p53 was confirmed by the experiments with a kinase-inactive Chk1. Only in the presence of an inducer molecule was phosphorylation of p53 at Ser-15 in the stable clones induced. Furthermore, its assay performance proved acceptable for high-throughput screening applications, judging from the Z' factor values (> 0.77). Finally, the cell-based assay thus established yielded structure-activity relationship data for a small set of test inhibitors of Chk1 within cells. Collectively, these results demonstrate that the established cell-based assay provides a novel and highly sensitive cellular platform for Chk1 inhibitor discovery.