Reversal of Fibrosis in Chronic Rejection in Mouse Airways through Combined Relaxin and Lysyl Oxidase Inhibitor Therapy

Abstract Background: Glaucoma is a neurodegenerative disease with the progressive loss of retinal ganglion cells and changes in the optic nerve head (ONH). These changes are exacerbated by an increase in intraocular pressure (IOP). Methods: The effect of scleral and optic nerve softening with beta aminopropionitrile a lysyl oxidase inhibitor (BAPN) and stiffening with genipin, in a model of chronic increase of IOP was evaluated. Changes in optic nerve and retina were evaluated. H&E, Bielschowsky's silver staining and glial fibrillary acid protein (GFAP) staining was performed on optic nerve, retina and scleral structures. Changes in the expression of the Ywhab, Yhwaz (prosurvival genes), C3 complement (complement C3 inflammatory marker), CPG15 (neurite growth and neural survival gene) GFAP (glial activator marker) genes was carried out in the different groups.Results: Protective effect of BAPN was evident by the preservation of the optic nerve structure, and with the conservation of the retinal structures, while deleterious changes were evident in the stiffening of ONH complex, characterized by the increase in the glia, changes in the optic nerve, and disorganization in the retina. BAPN induced a reduction in the expression of Ywhab, Yhwaz (prosurvival genes), C3 and GFAP (inflammatory and glial marker) and CPG15. Conclusions: These findings support the critical involvement of changes in the ONH stiffness in the progression of glaucoma. The control of this variable as a regulatory mechanism in the progression of neural glaucomatous damage must be considered and would be explored as a possible intervention in glaucoma management.

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Expression and properties of lysyl oxidase from archeal halophile Haloterrigena turkmenica

10.7287/peerj.preprints.2691v1 ◽

2017 ◽

Author(s):

Nikolay B Pestov ◽

Daniel V Kalinovsky ◽

Tatyana D Larionova ◽

Alia Z Zakirova ◽

Nikolai N Modyanov ◽

...

Keyword(s):

Catalytic Activity ◽

Amine Oxidase ◽

Lysyl Oxidase ◽

Polyclonal Antibodies ◽

Oxidase Inhibitor ◽

High Yield ◽

Transfer Event ◽

Oxidase Gene ◽

E Coli ◽

Haloterrigena Turkmenica

Background: Lysyl oxidases (LOX) were studied mostly in mammals, whereas properties of recently found homologs in prokaryotic genomes remain enigmatic. Methods: LOX gene from Haloterrigena turkmenica has been cloned by PCR in a E. coli expression vector. Protein purification has been done using metal affinity chromatography under denaturing conditions followed by refolding. Catalytic activity has been fluorometrically a release of hydrogen peroxide coupled with the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine in the presence of horseradish peroxidase. Rabbit polyclonal antibodies were obtained and used in western blotting. Results: H. turkmenica LOX (HTU-LOX) may be successfully expressed in E. coli with a high yield. However, full-length protein gives no catalytic activity. On the other hand, a deletion of putative signal peptide allows the protein to be refolded into an active enzyme. Further deletion until the boundary of the catalytic C-terminal domain greatly increases the activity. Refolding is optimal at pH around 6.0, with addition of Cu2+, and surprisingly does not respond to changing concentration s of NaCl. The active HTU-LOX is sensitive to the lysyl oxidase inhibitor β-aminopropionitrile. HTU-LOX is active towards usual substrates of mammalian LOX such as lysine-containing basic peptides and polymers. The major difference between HTU-LOX and mammalian LOX is a relaxed specificity of the former. HTU-LOX readily oxidizes various amines including such compounds as taurine and glycine. Moreover, it is active also towards aminoglycoside antibiotics. Benzyl amine is a poor substrate for HTU-LOX. H. turkmenica cells or culture medium do not contain any detectable amine oxidase activity. Polyclonal antibodies against HTU-LOX detect a band among H. turkmenica proteins with the molecular weight corresponding to the unprocessed enzyme. Conclusion: H. turkmenica contains a lysyl oxidase gene that may give active recombinant enzyme with important biochemical features conserved, for example, sensitivity to β-aminopropionitrile. However, its function in the host may be cryptic. Significance: This is the first report on some properties of lysyl oxidase that originated from a horizontal transfer event into Archea.

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