Reduction of the toxicity and mutagenicity of aziridine in mammalian cells harboring the Escherichia coli fpg gene.

Aziridine (ethyleneimine) reacts with DNA in vitro, mainly at the N7 position of guanine and N3 of adenine, then imidazole ring opening of the modified guanine results in formation of formamidopyrimidine (FaPy) residues. The Escherichia coli fpg gene encodes a DNA glycosylase that removes FaPy residues from DNA. To determine whether aziridine produces FaPy lesions in mammalian cells we have expressed the E.coli fpg gene in CHO cells. The transfected cells, expressing high levels of the bacterial protein, are more resistant to the toxic and mutagenic effects of aziridine than the control population. Less DNA damage was measured by quantitative PCR analysis in transfected than in control cells treated with equimolar concentrations of aziridine. The results suggest that aziridine produces in vivo FaPy residues that could account for the deleterious effects of this compound.     

9-[(10-(aden-9-yl)-4,8-diazadecyl)amino]-6-chloro-2-methoxy-acridine incises DNA at apurinic sites.

The incision of DNA at apurinic/apyrimidinic sites (AP-sites) by chloro-6-methoxy-2 [(adenyl-9)-11)-4,8 diazadecyl]amino-9 acridine (Ade-Z-Acr), a 9-aminoacridine linked to an adenine, at nanomolar concentrations is described. Moreover, this drug, Ade-Z-Acr, is one of the most efficient drugs which cleaves DNA at AP-sites. The high activity is the result of the composition of the drug, since the individual components have no incising activity in the concentration range studied. The termini left by the Ade-Z-Acr molecule are a 3'deoxyribose and a 5'nucleotide. The termini and the inability of the Ade-Z-Acr to incise DNA with reduced AP-sites suggest that the mechanism of cleavage is beta-elimination.     

Biochemical characterization and surfactant properties of horse allergens.

A new allergen from horse dander, Equ c 5 has been purified. Its biochemical and biophysical properties have been characterized and compared with those of Equ c 1, Equ c 2 and Equ c 4. Their molecular masses, determined by mass spectrometry, were 22 kDa for Equ c 1, 16 kDa for Equ c 2, 18.7 kDa for Equ c 4 and 16.7 kDa for Equ c 5. Their pI values were between 3.8 and 5.25. Equ c 2 and Equ c 5 are not glycosylated, while Equ c 4 contains a tri-antennary tri-sialylated N-linked glycan. Linkages of terminal N-acetylneuraminic acid to galactose were: alpha-(2-->6) in Equ c 4, and both alpha-(2-->3) and alpha-(2-->6) in Equ c 1. Oligosaccharide portions of Equ c 1 or Equ c 4 were barely involved in IgE-immunoreactivity. Partial N-terminal sequence of Equ c 4 shares a significant sequence homology with the rat submandibular gland protein A. No matching was found for two internal peptides of Equ c 5. Surfactant properties of horse allergens as well as other proteins were investigated. In contrast to Equ c 2 and Equ c 3, solutions of Equ c 1, Equ c 4 and Equ c 5 significantly lowered the surface tension. Relationship between a property such as this, involving oriented hydrophobic patches of a molecule and allergenicity, is addressed.     

1,N(2)-ethenoguanine,a mutagenic DNA adduct,is a primary substrate of Escherichia coli mismatch-specific uracil-DNA glycosylase and human alkylpurine-DNA-N-glycosylase

The promutagenic and genotoxic exocyclic DNA adduct 1,N(2)-ethenoguanine (1,N(2)-epsilonG) is a major product formed in DNA exposed to lipid peroxidation-derived aldehydes in vitro. Here, we report that two structurally unrelated proteins, the Escherichia coli mismatch-specific uracil-DNA glycosylase (MUG) and the human alkylpurine-DNA-N-glycosylase (ANPG), can release 1,N(2)-epsilonG from defined oligonucleotides containing a single modified base. A comparison of the kinetic constants of the reaction indicates that the MUG protein removes the 1,N(2)-epsilonG lesion more efficiently (k(cat)/K(m) = 0.95 x 10(-3) min(-1) nm(-1)) than the ANPG protein (k(cat)/K(m) = 0.1 x 10(-3) min(-1) nm(-1)). Additionally, while the nonconserved, N-terminal 73 amino acids of the ANPG protein are not required for activity on 1,N(6)-ethenoadenine, hypoxanthine, or N-methylpurines, we show that they are essential for 1,N(2)-epsilonG-DNA glycosylase activity. Both the MUG and ANPG proteins preferentially excise 1,N(2)-epsilonG when it is opposite dC; however, unlike MUG, ANPG is unable to excise 1,N(2)-epsilonG when it is opposite dG. Using cell-free extracts from genetically modified E. coli and murine embryonic fibroblasts lacking MUG and mANPG activity, respectively, we show that the incision of the 1,N(2)-epsilonG-containing duplex oligonucleotide has an absolute requirement for MUG or ANPG. Taken together these observations suggest a possible role for these proteins in counteracting the genotoxic effects of 1,N(2)-epsilonG residues in vivo.     

Differential acquisition of antigenic peptides by Hsp70 and Hsc70 under oxidative conditions

Hsp70 and Hsc70 are two chaperones of high homology expressed under contrasting situations. Hsc70 is constitutively expressed and poorly stress-inducible, whereas Hsp70 is unabundant in normal physiological situations and strongly induced under oxidative stress. In the present study we show that the chaperoning activity of purified Hsp70 and Hsc70 is minimal under reducing conditions and increases in environments that mimic oxidative stress. Association with peptides is more pronounced for Hsp70 than for Hsc70 in every condition tested and is accompanied with a gradual change in secondary structure during oxidation. The binding of peptides to Hsp70 and Hsc70 under oxidative conditions is not reversible by treatment with a reducing agent, confirming that other chaperone-associated factors are required for substrate release. These findings support the idea that formation of HSP70-peptide complexes and possibly their immunogenicity is enhanced in conditions of stress.     

Ruffini endings are absent from the periodontal ligament of trkB knockout mice

To clarify the role of neurotrophin receptors in the development of Ruffini endings, periodontal ligaments and trigeminal ganglia of trkA, trkB, and trkC knockout mice were immunostained for protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP), parvalbumin (PV), and calretinin (CR). Innervation patterns of PGP 9.5- and CGRP-immunoreactive fibers were examined in the periodontal ligament of the knockout mice. PGP 9.5-positive fibers in the incisal periodontal ligaments of trkA and trkC knockout mice form Ruffini endings distinguished by dendritic ramifications and branches. However, Ruffini endings were not present in the periodontal ligament of trkB knockout mice. Only free nerve endings were observed in tissue of trkB knockout mice. Compared with trkA and trkC knockouts, the proportion of CR-positive neurons in mandibular and maxillary regions of the trigeminal ganglion of trkB knockout mice is decreased. These findings indicate that the development of periodontal Ruffini endings is regulated by trkB-dependent and CR-coexpressing neurons.     

Inappropriate annotation of a key defence marker in<Emphasis Type="Italic"> Arabidopsis</Emphasis>: will the real<Emphasis Type="Italic"> PR-1</Emphasis> please stand up?

PR-1 has been extensively used as a marker for salicylic acid (SA)-mediated defence and systemic and local acquired resistance. The Arabidopsis Genome Project annotates At2g19990 as PR-1. This gene is also identified as PR-1 in two full genome Arabidopsis microarrays, and TAIR cites approximately 60 articles to describe its patterns of expression. However, most of these citations are incorrect; the probes used were not At2g19990, but a homologous gene At2g14610, which is annotated as PR-1-like. Because of the potential for confusion, we analyzed the expression of both genes in Arabidopsis thaliana (L.) Heynh. At2g14610 (PR-1-like) showed the archetypal patterns of SA-responsive expression: mRNA levels increased following SA-treatment, inoculation with an avirulent (but not a virulent) strain of Pseudomonas syringae, and in wild-type (but not NahG) Arabidopsis infected with cauliflower mosaic virus (CaMV). In cpr5 mutants it was expressed constitutively. In contrast, expression of At2g19990 (annotated as PR-1) was detectable in neither SA-treated Col-0 nor in cpr5. Infection by virulent and avirulent isolates of P. syringae up-regulated expression, but to a similar level, and infection by CaMV induced a modest increase in expression in both the wild type and NahG. At2g19990, although pathogen responsive, does not show the SA-dependent patterns of expression expected from a member of the PR-1 regulon, and its annotation as PR-1 is inappropriate. The annotations should identify At2g14610 as the authentic PR-1.     

Discovery of thiophene-2-carboxylic acids as potent inhibitors of HCV NS5B polymerase and HCV subgenomic RNA replication. Part 1: Sulfonamides

The discovery of a novel class of HCV NS5B polymerase inhibitors, 3-arylsulfonylamino-5-phenyl-thiophene-2-carboxylic acids is described. SAR studies have yielded several potent inhibitors of HCV polymerase as well as of HCV subgenomic RNA replication in Huh-7 cells.     

Functional Characterisation of Three O-methyltransferases Involved in the Biosynthesis of Phenolglycolipids in Mycobacterium tuberculosis

Phenolic glycolipids are produced by a very limited number of slow-growing mycobacterial species, most of which are pathogen for humans. In Mycobacterium tuberculosis, the etiologic agent of tuberculosis, these molecules play a role in the pathogenicity by modulating the host immune response during infection. The major variant of phenolic glycolipids produced by M. tuberculosis, named PGL-tb, consists of a large lipid core terminated by a glycosylated aromatic nucleus. The carbohydrate part is composed of three sugar residues, two rhamnosyl units and a terminal fucosyl residue, which is per-O-methylated, and seems to be important for pathogenicity. While most of the genes responsible for the synthesis of the lipid core domain and the saccharide appendage of PGL-tb have been characterized, the enzymes involved in the O-methylation of the fucosyl residue of PGL-tb remain unknown. In this study we report the identification and characterization of the methyltransferases required for the O-methylation of the terminal fucosyl residue of PGL-tb. These enzymes are encoded by genes Rv2954c, Rv2955c and Rv2956. Mutants of M. tuberculosis harboring deletion within these genes were constructed. Purification and analysis of the phenolglycolipids produced by these strains, using a combination of mass spectrometry and NMR spectroscopy, revealed that Rv2954c, Rv2955c and Rv2956 encode the methyltransferases that respectively catalysed the O-methylation of the hydroxyl groups located at positions 3, 4 and 2 of the terminal fucosyl residue of PGL-tb. Our data also suggest that methylation at these positions is a sequential process, starting with position 2, followed by positions 4 and 3.