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Author(s): Guirong Li [sup.1] [sup.2], Chenyou Shen [sup.1], Dong Wei [sup.3], Xusheng Yang [sup.1], Cheng Jiang [sup.1], Xiucheng Yang [sup.3], Wenjun Mao [sup.4], Jian Zou [sup.2], Jianxin Tan [sup.1], Jingyu [...], Purpose Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease characterized by excessive extracellular matrix deposition. No effective treatments are currently available for IPF. High-temperature requirement A3 (HtrA3) suppresses tumor development by antagonizing transforming growth factor [beta] (TGF-[beta]) signaling; however, little is known about the role of HtrA3 in IPF. This study investigated the role of HtrA3 in IPF and underlying mechanisms. Methods Lung tissues were collected from patients with IPF and mice with bleomycin (BLM)-induced pulmonary fibrosis, and HtrA3 expression was measured in tissue samples. Then, HtrA3 gene knockout mice were treated with BLM to induce pulmonary fibrosis and explore the effects and underlying mechanism of HtrA3 on pulmonary fibrosis. Results HtrA3 was up-regulated in the lung tissues of patients with IPF and the pulmonary fibrotic mouse model compared to corresponding control groups. HtrA3 knockout decreased pulmonary fibrosis-related protein expression, alleviated the symptoms of pulmonary fibrosis, and inhibited epithelial-mesenchymal transition (EMT) in BLM-induced lung tissue compared with BLM-induced wild-type mice. The TGF-[beta]1/Smad signaling pathway was activated in fibrotic lung tissue, whereas HtrA3 knockout inhibited this pathway. Conclusion The expression level of HtrA3 is increased in fibrotic lungs. HtrA3 knockout alleviates the symptoms of pulmonary fibrosis probably via the TGF-[beta]1/Smad signaling pathway. Therefore, HtrA3 inhibition is a potential therapeutic target for pulmonary fibrosis.

Research discusses a structural model for the mechanism of a second cleavage event in the antitumor antibiotic bleomycin. The model demonstrates a bithiazole tail of the antibiotic partially intercalating between the T19 and the A20 dihedral angles of the duplex DNA.

Results demonstrate that ferric bleomycin and ferrous bleomycin bind to DNA using their adducts analogous to the cobalt bleomycin species binding but different from that of porphyrins. Data indicate that restrictive effect of DNA intercalation with the bithiazole tail of the bleomycin may be involved in this differential binding pattern.

A study was conducted to analyze the structure and reactivity of iron complexes of PMAH, a synthetic analogue of bleomycin supporting a structure that mimics the metal chelating domain of the drug. The ferric complex supporting the electron paramagnetic resonance spectrum was determined. Results suggested that iodosylbenzene-mediated oxotransfer reactions may occur without the formation of a hypervalent metal intermediate.

A study was conducted to examine the expression of extracellular matrix glycoprotein tenascin (TN) during pulmonary injury brought about by intratracheal bleomycin. Rat lung tissues were deparaffinized, rehydrated and digested with trypsin while some sections of the rat lungs underwent in situ hybridization with 35S-cRNA. Results confirmed TN as an early-response extracellular matrix component to bleomycin-induced pulmonary injury.

A study surveyed the metal domain properties of Fe- and Co-bleomycin species in the presence of two DNA decamers, one of which contained a specific site of binding while the other, related one had its 5'-AT-3' substituted for 5'-GC-3', d(GGAAATTTCC)2 (DNAb). Findings support the hypothesis that when these metallobleomycin species bind to DNA, the metal domain can adopt two different conformations and these conformations depend on whether a specific site of binding is present. Experimental methods and results are presented.

NMR studies of the paramagnetic Fe(II)BLM identified the ligands to the metal center in the Fe(II) adduct of bleomycin as derived from the beta-aminoalanine, the pyrimidinylpropionamide and the beta-hydroxyhistidine segments. Two-dimensional NMR techniques also allowed the identification of many of the spin networks present in the NMR spectrum of the paramagnetic Fe(II)BLM. Moreover, they clarify the assignments for the signals between -0.8 and -20 ppm. Examination of the NMR data reveals that alpha-methylvalerate and/or mannose segments do not participate in Fe(II) complexation.

The structure of activated and reactive bleomycin measured by electron nuclear double resonance (ENDOR) method shows hyperfine coupling due to structural anisotropy and quadrapolar coupling. Consideration of one strongly coupled oxygen gives good agrement between the results of ENDOR and electron paramagnetic resonance studies. The largest hyperfine coupling of exchangeable proton and well-resolved line along the g(sub z) axis suggest that this direction is responsible for the ligation of amine and oxygen ligands which can form hydrogen bonds.

Bleomycin A(sub 2) can act as the medium for the oxidative cleavage of double-stranded DNA or RNA by a metal ion and oxygen dependant chemical process. The metal binding domain of the bleomycin A(sub 2) in the full natural product structure controls the selective determination of DNA cleavage although the presence of noncovalently linked tri-and tetrapeptide S cannot show the sequence selective DNA cleavage. The metal binding domain of bleomycin A(sub 2) consists of iron (II) and iron (III) complexes.

The key synthetic analogs of bleomycin A(sub 2) 2-O-(3-O-carbamoyl)-alfa-D-mannopyranoside has the same DNA cleavage efficiency, selectivity, and double strand versus single strand DNA cleavage ratio showing that the DNA cleavage capabilities of the natural agent are uninfluenced by carbonyl groups. The DNA cleavage efficiency, the rate of single strand versus double strand DNA cleavage ratio of demannosyslbleomycin A(sub 2) and deglycobleomycin A(sub 2) shows the DNA cleavage capability of the natural agent is highly affected by the first carbohydrate of the disaccharide.

Analysis of the substrate formed by Escherichia coli 5S ribosomal RNA's reverse transcription reveals the effect of iron(II) bleomycin on the DNA-RNA heteroduplex. Fe(II).BLMA2 cleaves the two strands of the heteroduplex at ambient concentrations and application of 5 micromol of Fe(II)BLMA2 completely digests both strands. The heteroduplexes may function as targets for bleomycin when there is excess physiological concentrations of doubly ionized magnesium ions.

The binding properties of ferrous bleomycin and dioxygen in the presence of DNA reveals that there is a high spaced interaction of Fe(II)Blm.DNA. The high speed interaction forms an adduct which is oxidation stable. The stability of the adduct increases basepair ratio of the drug.

An analysis of the first total synthesis of (+)-P-3A (1), epi-(-)-P-3A (2) and (-)-desacetamido P-3A (3) confirmed that Fe(II) complexes of 1, 2 and 3 cleave duplex DNA above background Fe(II) almost as well as deglycobleomycin A2, without any noticeable sequence selectivity. A bleomycin-linked metal chelation subunit is inadequate to provide evidence of the typical DNA cleavage selectivity.

V. Martins (1); D. B. Lopes (1); W. R. Teodoro (2); V. L. Capelozzi. (1) (1) Department of Pathology and (2) Rheumatology Discipline, Faculty of Medicine of University of Sao [...], Context: It is well known that the initiation, distribution, and severity of fibrosis vary among different chemical injuries. However, the mechanisms of chemical variation and their epithelial/mesenchymal basis, as well as the activation of susceptibility to pulmonary fibrosis by profibrotic signals, are poorly investigated. Because of this, we aimed to evaluate and compare the activation profile and perpetuation of fibrosis induced by bleomycin (BLM) and 3-5-di-ieri-4-hydroxytoluene (BHT). Design: For pulmonary fibrosis, groups of male mice (C57Bl/6; n = 6) had BLM (0.1 U) administered by an intratracheal or intraperitoneal route with BHT (400 mg). Sterile saline or corn oil was used as control. Lungs were removed 14 (early) and 21 (late) days after induction, and analysis was performed. All experimental procedures were performed according to the guidelines of the Ethical Committee of the Faculty of Medicine of University of Sao Paulo, Brazil (process code 372/11). Results: Higher amounts of collagen, evaluated by hydroxyproline and Masson staining, were found in BLM and BHT groups when compared to controls (P Conclusions: The upregulation of cytokines and growth factors resulted in an increase of total and specific types of lung collagen accumulation in the lungs of BLM and BHT groups. However, distinct mechanisms of peribronchiolar and peripheral fibrosis may be involved, since we found down-regulation of the AGT gene in the late BHT group. Financial support: FAPESP.

Experiments aimed at analyzing the effect of bleomycin on alveolar macrophages-induced gelatinase release helped understand the role of gelatinase products from macrophages in the bleomycin-based model representing pulmonary fibrosis. Zymograms which comprise gelatin or collagen substrates provided the surface for the analysis of enzyme activity of 92-KDa gelatinase. Western blot analysis elucidated the homology between gelatinase and the metalloproteinase released by rat mammary carcinoma cells.

H NMR titration experiments demonstrate that the conventional DNA-binding domain of bleomycin effectively bound to the L-enantiomer. This was researched by employing L-d(CGCGCG), the enantiomer of natural D-d(CGCGCG). The chirality of DNA determined the behavior exhibited by the other linker moiety and metal-binding domain. The linker-metal-binding zone mainly determines the specific DNA recognition of bleomycin.

The resonance Raman data that identify two important vibrational modes in the Co-OOh linkage that is the stretching modes of v(Co-OOH) and v(O-OH), is depicted. The task of the high-frequency resonance Raman vibration modes for HOO-Co(III)Blm and Co(III)Blm strongly supports an isostructural relationship between FeBlm and CoBlm.

Kinetic studies of oxygen isotope effect on activated bleomycin prepared from either natural abundance O2 or 97 atom % 18O2 reveal that the rate of activated bleomycin decay is 1.054 (plus or minus) 0.008. Kinetic isotope effects and relative sizes of equilibrium help understand the structure of the transition state. Formation of activated bleomycin precedes the O-O cleavage, as revealed by the presence of peroxide in the activated bleomycin. An unstable monooxygenated species generated from the activated bleomycin is the major DNA-attacking species.

An analysis directed at the DNA strand scission by photoactivated promazine derivatives established three chlorinated bithiazole derivatives which, at very low concentrations, mediate light dependent DNA cleavage. These derivatives are structurally associated with bleomycin A5. The N-t-Boc derivative of methyl 2'-(2-aminoethyl)-2, 4'-bithiazole-4-carboxylate formed the starting point for the synthesis of bithiazoles.