Mutagenesis in cultured human diploid cells. III. Induction of 8-azaguanine-resistant mutations by furylfuramide.

Trans-2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (furylfuramide: FF or AF2) was tested for ability to induce 8-azaguanine (8AG) resistant mutations in cultured human diploid cells. FF had a relatively severe cytotoxic effect on the cells. From the concentration-survival curve, the D0 value for 2-h treatment with FF was estimated to be 11 mug/ml. When cells were treated with FF at various concentrations for 2 h, cultured in normal medium for 48 h, and then selected in medium containing 8AG at 30 mug/ml, the induced mutation frequency increased gradually with increase in concentration of FF. When cells were treated with FF at 10 mug/ml for 2 h, cultured in normal medium for various periods of mutation expression time, and selected with 8AG at 30 mug/ml, the highest induced mutation frequency was obtained with 48 h of mutation expression time. Microscopic examination of the numbers of cells in colonies indicated that the total number of cells increased by half during this mutation expression time of 48 h.     

Src tyrosine kinase inhibitor PP2 suppresses ERK1/2 activation and epidermal growth factor receptor transactivation by X-irradiation

Exposure of MDA-MB-468 cells to ionizing radiation (IR) caused biphasic activation of ERK as indicated by its phosphorylation at Thr202/Tyr204. Specific epidermal growth factor receptor (EGFR) inhibitor AG1478 and specific Src inhibitor PP2 inhibited IR-induced ERK1/2 activation but phosphatidylinositol-3 kinase inhibitor wortmannin did not. IR caused EGFR tyrosine phosphorylation, whereas it did not induce EGFR autophosphorylation at Tyr992, Tyr1045, and Tyr1068 or Src-dependent EGFR phosphorylation at Tyr845. SHP-2, which positively regulates EGFR/Ras/ERK signaling cascade, became activated by IR as indicated by its phosphorylation at Tyr542. This activation was inhibited by PP2 not by AG1478, which suggests Src-dependent activation of SHP-2. Src and PTPalpha, which positively regulates Src, became activated as indicated by phosphorylation at Tyr416 and Tyr789, respectively. These data suggest that IR-induced ERK1/2 activation involves EGFR through a Src-dependent pathway that is distinct from EGFR ligand activation.     

Antigen 85C-mediated acyl-transfer between synthetic acyl donors and fragments of the arabinan

Antigen 85 (ag85) is a complex of acyltransferases (ag85A–C) known to play a role in the mycolation of the d-arabino-d-galactan (AG) component of the mycobacterial cell wall. In order to better understand the chemistry and substrate specificity of ag85, a trehalose monomycolate mimic p-nitrophenyl 6-O-octanoyl-β-d-glucopyranoside (1) containing an octanoyl moiety in lieu of a mycolyl moiety was synthesized as an acyl donor. Arabinofuranoside acceptors, methyl α-d-arabinofuranoside (2), methyl β-d-arabinofuranoside (3), and methyl 2-O-β-d-arabinofuranosyl-α-d-arabinofuranoside (9) were synthesized to mimic the terminal saccharides found on the AG. The acyl transfer reaction between acyl donor 1 and acceptors 2, 3, and 9 in the presence of ag85C from Mycobacterium tuberculosis (M. tuberculosis) resulted in the formation of esters, methyl 2, 5-di-O-octanoyl-α-d-arabinofuranoside (10), methyl 5-O-octanoyl-β-d-arabinofuranoside (11), and methyl 2-O-(5-O-octanoyl-β-d-arabinofuranosyl)-5-O-octanoyl-α-d-arabinofuranoside (12) in 2 h, 2 h and 8 h, respectively. The initial velocities of the reactions were determined with a newly developed assay for acyltransferases. As expected, the regioselectivity corresponds to mycolylation patterns found at the terminus of the AG in M. tuberculosis. The study shows that d-arabinose-based derivatives are capable of acting as substrates for ag85C-mediated acyl-transfer and the acyl glycoside 1 can be used in lieu of TMM extracted from bacteria to study ag85-mediated acyl-transfer and inhibition leading to the better understanding of the ag85 protein class. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

LPS-stimulated MUC5AC production involves Rac1-dependent MMP-9 secretion and activation in NCI-H292 cells

Chronic obstructive pulmonary disease (COPD) is an inflammatory process characterized by airway mucus hypersecretion. Previous studies have reported that lipopolysaccharides (LPS) stimulate mucin 5AC (MUC5AC) production via epidermal growth factor receptor (EGFR) in human airway cells. Moreover, this production was shown to depend on the expression and activity of matrix metalloproteinase 9 (MMP-9), which is increased in COPD patients’ serum. In the present study we investigated the signaling pathway mediating LPS-stimulated secretion and activation of MMP-9, and the regulatory effects of this pathway on the production of MUC5AC in the human airway cells NCI-H292. Using specific inhibitors, we found that LPS-stimulated cells secreted and activated MMP-9 via EGFR. Our results also indicate that signaling events downstream of EGFR involved PI3K-dependent activation of Rac1, which mediated the NADPH-generated reactive oxygen species responsible for MMP-9 secretion and activation. Finally, we observed that EGFR/PI3K/Rac1/NADPH/ROS/MMP-9 regulate MUC5AC production in LPS-challenged NCI-H292 cells.     

Binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against SARS

In order to stimulate the development of drugs against severe acute respiratory syndrome (SARS), based on the atomic coordinates of the SARS coronavirus main proteinase determined recently [Science 13 (May) (2003) (online)], studies of docking KZ7088 (a derivative of AG7088) and the AVLQSGFR octapeptide to the enzyme were conducted. It has been observed that both the above compounds interact with the active site of the SARS enzyme through six hydrogen bonds. Also, a clear definition of the binding pocket for KZ7088 has been presented. These findings may provide a solid basis for subsite analysis and mutagenesis relative to rational design of highly selective inhibitors for therapeutic application. Meanwhile, the idea of how to develop inhibitors of the SARS enzyme based on the knowledge of its own peptide substrates (the so-called "distorted key" approach) was also briefly elucidated.     

DNA crosslinking, sister-chromatid exchange and specific-locus mutations

Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds.     

Mutagenic effect of orally given AF-2 on embryonic cells in pregnant Syrian hamsters

Pregnant hamsters were given various doses of AF-2 by stomach tube; then the cells of their embryos were isolated and cultured in normal medium. Chromosome preparations were made within 24 h after the start of primary culture, and examined for chromosomal aberrations. Marked chromosomal abnormalities were observed in cells of embryos of animals treated with AF-2 at over 20 mg/kg. Samples of surviving cells were also cultured in normal medium for 48 h, and then selected in medium containing 8AG or 6TG. This treatment with AF-2 caused marked dose-dependent induction of 8AG- or 6TG-resistant mutations: mutant colonies were even obtained after a single treatment with 2 mg of AF-2 per kg. These results show that this is a sensitive and useful mammalian system for detecting environmental mutagens.     

Mutagenesis by simian virus 40. I. detection of mutations in Chinese hamster cell lines using different resistance markers.

The mutagenic action of SV40 in permanent lines of Chinese hamster cells (CHO-K1 and V79) was investigated with the aid of different resistance markers. The markers studied had resistance to 8-azaguanine (25 and 30 mug/ml), aminopterin (3.3--5.5X10(-3) mug/ml), colchicine (6.5 and 7.0X10(-2) mug/ml) and 5-bromodeoxyuridine (50--120 mug/ml), respectively. After virus infection the mutation frequencies were increased by one (azaguanine, aminopterin) and two (colchicine) orders of magnitude as compared with spontaneous mutation frequencies. In contrast, it was not possible to enhance the frequency of mutation to BUdR resistance. On the other hand, the ability to proliferate in HAT medium was induced in three of five BUdR-resistant cell clones by infection with SV40. The resistance induced by SV40 was stable when isolated clones were cultured under non-selective conditions. Mechanisms are proposed that may be responsible for the mutagenic action of SV40.     

The role of cuticle and epidermal cell wall in resistance of rapeseed and mustard to Rhizoctonia solani

A study was conducted to determine whether the cuticles in two genera of the family Cruciferae are effective barriers to infection by Rhizoctonia solani, and whether differences in cuticle and epidermal cell wall thickness and morphology of epicuticular wax exist between resistant and susceptible cultivars. As Canola/rapeseed (Brassica napus) and mustard (Sinapis alba) plants develop from 1 to 3 weeks of age, they become increasingly resistant to R. solani AG2-1 seedling root rot. Seven-day-old seedlings of S. alba cultivars are invariably more resistant than B. napus cultivars. Brassica napus cultivars do not show an obvious cuticle layer at 1 week but at 3 weeks the presence of a cuticle is seen through autofluorescence with a concomitant increase in resistance to R. solani. Removal of the cuticle from 3-week-old hypocotyls by chloroform treatment results in a decrease in cuticular autofluorescence and a significant increase in disease severity in both resistant and susceptible cultivars. Three-week-old plants of S. alba have a much lower percent disease rating and a significantly (p=0.05) thicker cuticle layer than similar-age plants of B. napus. The results suggest that the cuticle plays an important role in the resistance of S. alba and older plants of B. napus to infection by R. solani.