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.     

Does Pattern Scan Laser (PASCAL) photocoagulation really induce less VEGF expression in murine retina than conventional laser treatment?

To investigate the differences in the mRNA and protein expression levels of vascular endothelial growth factor (VEGF) in murine retina between mice subjected to conventional laser (AG) and those subjected to Pattern Scan Laser (PASCAL) system. Male C57BL/6 mice were randomly assigned to one of three groups: Group 1 (G1) receiving retinal scatter laser photocoagulation using with AG photocoagulator (n = 16), Group 2 (G2) receiving retinal scatter laser photocoagulation using with PASCAL (n = 16) and Group 3 (G3) served as an untreated control group (n = 6). Molecular and morphological analyses of VEGF were performed on days 1, 2 and 5 by ELISA, real-time PCR and immuno-histochemical analysis. In samples which underwent AG (G1), when compared with the control group (G3), VEGF mRNA level increased 2.4 folds on day 2, whereas it decreased on day 5 (p □ 0.001). In samples which underwent PASCAL (G2), on the other hand, VEGF mRNA level increased 1.8 folds on day 1 and 2.2 folds on day 5 when compared with the control group (G3). In samples which underwent AG (G1), when compared with the control group (G3), VEGF protein level increased significantly on day 2, whereas it decreased on day 5 (p □ 0.001). In group G2, the VEGF levels in the sensory retina significantly increased as compared to control groups at both 2 and 5 days after laser photocoagulation using PASCAL laser (p = 0.012, both time points). The peak expressions of VEGF protein in samples which underwent PASCAL and conventional laser were found on day 5 and day 2 respectively. In retinas of PASCAL-treated mice, VEGF immunoreactivity gradually increased during the 5-day follow-up. However, in argon laser group, the strongest VEGF immunoreactivity was detected on day 2, then started to decrease on day 5. In summary, the expression of VEGF protein and mRNA gradually increase during a 5-day follow-up period in PASCAL-treated mouse eyes, whereas in AG group they reach their peak levels on the second day of follow-up and started decreasing after then. These results may also explain why the PASCAL system is less effective in regressing neovascularization in the clinic.     

Recent advancements in the development of anti-tuberculosis drugs

Modern chemotherapy has significantly improved patient outcomes against drug-sensitive tuberculosis. However, the rapid emergence of drug-resistant tuberculosis, together with the bacterium’s ability to persist and remain latent present a major public health challenge. To overcome this problem, research into novel anti-tuberculosis targets and drug candidates is thus of paramount importance. This review article provides an overview of tuberculosis highlighting the recent advances and tools that are employed in the field of anti-tuberculosis drug discovery. The predominant focus is on anti-tuberculosis agents that are currently in the pipeline, i.e. clinical trials.     

Tyrphostin AG556 increases the activity of large conductance Ca2+‐activated K+ channels by inhibiting epidermal growth factor receptor tyrosine kinase

The present study was designed to investigate whether large conductance Ca²⁺‐activated K⁺ (BK) channels were regulated by epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase. BK current and channel tyrosine phosphorylation level were measured in BK‐HEK 293 cells expressing both functional α‐subunits and the auxiliary β1‐subunits using electrophysiology, immunoprecipitation and Western blotting approaches, respectively, and the function of rat cerebral basilar arteries was determined with a wire myography system. We found that BK current in BK‐HEK 293 cells was increased by the broad spectrum protein tyrosine kinase (PTK) inhibitor genistein and the selective EGFR tyrosine kinase inhibitor AG556, one of the known tyrphostin. The effect of genistein or AG556 was antagonized by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. On the other hand, orthovanadate or EGF decreased BK current, and the effect was counteracted by AG556. The tyrosine phosphorylation level of BK channels (α‐ and β1‐subunits) was increased by EGF and orthovanadate, while decreased by genistein and AG556, and the reduced tyrosine phosphorylation of BK channels by genistein or AG556 was reversed by orthovanadate. Interestingly, AG556 induced a remarkable enhancement of BK current in rat cerebral artery smooth muscle cells and relaxation of pre‐contracted rat cerebral basilar arteries with denuded endothelium, and these effects were antagonized by the BK channel blocker paxilline or orthovanadate. These results demonstrate that tyrosine phosphorylation of BK channels by EGFR kinase decreases the channel activity, and inhibition of EGFR kinase by AG556 enhances the channel activity and dilates rat cerebral basilar arteries.     

Structural and catalytic insights into the algal prostaglandin H synthase reveal atypical features of the first non-animal cyclooxygenase

Prostaglandin H synthases (PGHSs) have been identified in the majority of vertebrate and invertebrate animals, and most recently in the red alga Gracilaria vermiculophylla. Here we report on the cloning, expression and characterization of the algal PGHS, which shares only about 20% of the amino acid sequence identity with its animal counterparts, yet catalyzes the conversion of arachidonic acid into prostaglandin-endoperoxides, PGG₂ and PGH₂. The algal PGHS lacks structural elements identified in all known animal PGHSs, such as epidermal growth factor-like domain and helix B in the membrane binding domain. The key residues of animal PGHS, like catalytic Tyr-385 and heme liganding His-388 are conserved in the algal enzyme. However, the amino acid residues shown to be important for substrate binding and coordination, and the target residues for nonsteroidal anti-inflammatory drugs (Arg-120, Tyr-355, and Ser-530) are not found at the appropriate positions in the algal sequences. Differently from animal PGHSs the G. vermiculophylla PGHS easily expresses in Escherichia coli as a fully functional enzyme. The recombinant protein was identified as an oligomeric (evidently tetrameric) ferric heme protein. The preferred substrate for the algal PGHS is arachidonic acid with cyclooxygenase reaction rate remarkably higher than values reported for mammalian PGHS isoforms. Similarly to animal PGHS-2, the algal enzyme is capable of metabolizing ester and amide derivatives of arachidonic acid to corresponding prostaglandin products. Algal PGHS is not inhibited by non-steroidal anti-inflammatory drugs. A single copy of intron-free gene encoding for PGHS was identified in the red algae G. vermiculophylla and Coccotylus truncatus genomes.     

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.     

Molecular characterisation and expression of two venom allergen-like protein genes in Heterodera glycines.

Secretory proteins encoded by genes expressed in the oesophageal gland cells of plant-parasitic nematodes have key roles in nematode parasitism of plants. Two venom allergen-like protein cDNAs (designated hg-vap-1 and hg-vap-2)were isolated from Heterodera glycines gland cell cDNA libraries. Both cDNAs hybridised to genomic DNA of H. glycines in Southern blots. The hg-vap-1 cDNA contained an open reading frame encoding 215 amino acids with the first 25 amino acids being a putative secretion signal. The hg-vap-2 cDNA contained an open reading frame encoding 212 amino acids with the first 19 amino acids being a putative secretion signal. Genes of hg-vap-1 and hg-vap-2 contained four introns, which ranged in size from 44 to 574 bp, and five exons ranging in size from 43 to 279 bp. In situ hybridisation analyses showed that mRNAs of both vap genes accumulated specifically in the subventral gland cells of H. glycines during parasitism. The gland cell-specific expression and presence of predicted secretion signal peptides in both VAPs suggest that these proteins are secreted from the nematode and may play a role in the infection of host plants by this parasite.     

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.     

Contents Volume 93 (1982)

A thymidine kinase heterozygote was isolated from a diploid human lymphoblast line which forms colonies with high efficiency in microtiter dishes. We show that this cell line, called TK6, can be mutated from a tK^+/? to TK^?/? state by diverse mutagens, including ethyl methanesulfonate, butyl methanesulfonate, nitrosomethylurea, UV light, ICR-191, 4-nitroquinoline oxide, fluorodeoxyuridine, benzo[a]pyrene and aflatoxin B₁. We report here the experiments required to demonstrate the applicability of this new line in quantitative assays of mutation in human cells.Mitotic recombination between the centromere and the tk locus could not be induced by either dimethylsulfoxide or phorbol-12-myristate-13-acetate.