Phorbol ester facilitates apoptosis in murine fibroblasts pretreated by mild ultraviolet radiation.

Although phorbol 12-myristate 13-acetate (PMA) inhibits apoptosis and promotes the growth of some types of cells, it induces apoptosis in other cells. We evaluated the apoptotic effects of PMA on murine fibroblasts (L-929) that had been exposed to ultraviolet-B (UV-B) radiation at 312 nm, which promotes tumor cell growth. Exposure to PMA alone did not induce Fas, Fas-L, or apoptosis. Cells exposed to mild UV-B irradiation (80 J/m(2)) alone exhibited a slight expression of Fas and Fas-L 36 to 48 h after the exposure, and exhibited apoptosis as evidenced by DNA fragmentation 72 h after exposure. The addition of PMA (0.8 x 10(-5) to 3.2 x 10(-5) M) to the medium 24 h after the UV-B exposure markedly and dose-dependently enhanced these cell responses. Confluent untreated cells, cells cocultured with PMA, and cells cocultured with PMA for 24 h after the UV-B exposure consistently expressed mRNAs for wild-type p53, bcl-2, and ICE. Expression of c-myc mRNA was initially observed, but became undetectable in the cells cocultured for 24 h with a high concentration of PMA (3.2 x 10(-5) M) following UV-B exposure. Such cells subsequently exhibited the maximal apoptotic response. We conclude that mild exposure to UV-B altered murine fibroblast cells in such a way as to facilitate their death by apoptosis upon addition of PMA.     

Monoclonal antibodies against beta-galactoside-binding lectin of chick embryo recognizes conformational change of the antigen

Monoclonal antibodies against chick embryonic beta-galactoside-binding lectin were obtained. One of the monoclonal antibodies was ineffective in Western blotting and seemed to be unable to bind the SDS-denatured lectin. When the native lectin was dotted on a nitrocellulose filter and subjected to denaturation by treatment with SDS, urea or heat, binding of this antibody no longer occurred, though other monoclonal antibodies bound normally. This antibody seems to have been raised against an epitope which is destroyed upon denaturation.     

Studies on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor and thermolysin.

The effects of certain physicochemical parameters on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor (SMPI) and thermolysin were investigated. SMPI had its lowest Ki value at a pH of around 6.5 (similar to the pH dependence of the kcat/K(m) of thermolysin catalysis), reflecting the splitting mechanism of the SMPI inhibition of thermolysin. This Ki increased with an increase in pressure, and in (Ki-1) was almost linear with respect to pressure. The volume of the reaction (delta Vcomp), which is the volume change accompanying enzyme-inhibitor complex formation, was calculated as +8.1 +/- 0.3 mL.mol-1, which has a sign opposite to delta Vcomp for neutral peptide inhibitors and acyl-peptide substrates. The temperature dependence of Ki-1 gave the reaction enthalpy (delta Hcomp) and reaction entropy (delta Scomp) of the complex formation as 34.6 +/- 1.4 kJ.mol-1 and 298 +/- 5 J.mol-1.K-1, respectively. These positive reaction volumes and reaction entropies were related to the electrostatic interactions and ionic strength dependence of Ki which corresponded to the key ionic interaction during complex formation. Complex formation with SMPI stabilized thermolysin against pressure perturbation as observed by the changes in the Trp fluorescence of thermolysin with increasing pressure. Thermal stability, however, was affected very little by complex formation with SMPI. Phosphoramidon, Cbz-Phe-Gly-NH2 and Cbz-Phe also positively affected the pressure-tolerance of thermolysin, in the following order: Cbz-Gly-Phe-NH2 < Cbz-Phe < phosphoramidon. The third compound exhibited stabilizing effects comparable with those of SMPI, which suggests that the interaction between SMPI and thermolysin was localized to the reactive site.     

Protein polymorphism in a feral population of the pigeon Columba livia domestica

Sixteen enzymatic and non-enzymatic proteins of the pigeon Columba livia domestica were examined electrophoretically. These proteins were presumed to be under control by 22 loci. Of the 22 loci, 6 were defined as polymorphic and 15 as monomorphic. Another locus was variable, but the variation was not genetically interpretable. Average heterozygosity calculated over 21 loci was 0.075.     

Involvement of the Golgi region in the intracellular trafficking of cholera toxin

The intracellular pathway following receptor-mediated endocytosis of cholera toxin was studied using brefeldin A (BFA), which inhibited protein secretion and induced dramatic morphological changes in the Golgi region. In both mouse Y1 adrenal cells and CHO cells, BFA at 1 μg/ml caused a 80–90% inhibition of the cholera toxin (CT)-elevation of intracellular cAMP. The inhibition of the cytotoxicity of CT by BFA was also observed in a rounding assay of Y1 adrenal cells. The inhibition of CT cytotoxicity by BFA was dose dependent, with the ID₅₀ value similar to the LD₅₀ of BFA in Y1 adrenal cells. Binding and internalization of [¹²⁵I]-cholera toxin in Y1 adrenal cells was not affected by BFA. Unlike the BFA-sensitive cell lines such as Y1 adrenal and CHO cells, BFA at 1 μg/ml did not inhibit the cytotoxicity of CT in PtK₁ cells, of which the Golgi structure was BFA-resistant. These results strongly suggest that a BFA-sensitive Golgi is required for the protection of CT cytotoxicity by BFA. In contrast, elevation of the intracellular cAMP by forskolin, which acts directly on the plasma membrane adenylate cyclase, was not affected by BFA. These observations indicate that the intoxication of target cells by CT requires an intact Golgi region for its intracellular trafficking and/or processing. In this respect, CT shares a common intracellular pathway with ricin, Pseudomonas toxin, and modeccin, even though their structures and modes of action are very different. © 1993 Wiley-Liss, Inc.     

Genetic analysis of a mutation affecting ribosomal protein S1 in Escherichia coli

Molecular Genetics and Genomics - Ribosomal protein S1 from a newly isolated Escherichia coli mutant has a molecular weight of about 54,000 which is smaller than the wild type S1 (M.W. 65,000). The...     

Phosphorylation sites of bovine brain myelin basic protein phosphorylated with Ca2+-calmodulin-dependent protein kinase from rat brain

The phosphorylation sites of myelin basic protein from bovine brain were determined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala- and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue.     

Internalization of serratial protease into cells as an enzyme-inhibitor complex with alpha 2-macroglobulin and regeneration of protease activity and cytotoxicity

Extracellular serratial protease (56,000 Da) is known to be cytotoxic. Fluorescein isothiocyanate-labeled protease was found to form a complex with human alpha 2-macroglobulin (alpha 2M), and this enzyme-inhibitor complex was purified. The protease was found to be internalized by fibroblasts in culture as a complex with alpha 2M, which resulted in cell destruction. Regeneration of enzyme activity was confirmed in cells after 2-3 h of incubation. Chicken egg-white ovomacroglobulin, a homolog of human alpha 2M, formed a complex with this enzyme similarly and more tightly but failed to exhibit protease activity, cytotoxicity, and internalization into cells.     

DNA strand breaks in mammalian tissues induced by methylarsenics

DNA damage induced by administration of dimethylarsinic acid (DMAA) to rats and mice was investigated. At 12 h after administration of DMAA, DNA single-strand breaks were induced markedly in lung. The majority of dimethylarsine, one of the main metabolites, in the expired air was excreted within 6–18 h after administration of DMAA to rats. In vitro experiments using nuclei isolated from lung of mice indicated that DNA strand breaks were caused by dimethylarsine. Furthermore, the strand breaks after exposure to dimethylarsine were reduced in the presence of catalase and/or superoxide dismutase. These results strongly suggest that the strand breaks are induced not by dimethylarsine itself but by active oxygen, e.g., O ₂ ^? and ·OH, produced both by dimethylarsine and molecular oxygen. When DNA was exposed to dimethylarsine, thiobarbituric acid (TBA)-reactive intermediates andcis-thymine glycol were produced. Dimethylarsine appears to induce DNA damage by the mechanism similar to the damage produced by ionizing radiation.