Anti-stress activity of Propionibacterium freudenreichii: identification of a reactivative protein

Propionibacterium freudenreichii subsp. shermanii is known to prevent mutations caused by various agents such as N-methyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, 4-nitro-quinoline-1-oxide and by UV radiation in both prokaryotic and eukaryotic cells. It was also shown to prevent or repair damage caused by H(2)O(2) or UV radiation in Salmonella typhimurium and Escherichia coli, a characteristic previously designated as reactivative effect. In order to characterise this effect at the molecular level, we have purified the active component from a P. freudenreichii cell-free extract using a combination of ammonium sulfate precipitation, anion-exchange and size-exclusion chromatography. The isolated 35 kDa protein was then identified using both N-terminal and internal peptide sequencing as a cysteine synthase. The latter was localised in the P. freudenreichii proteomic map. It is constitutively expressed but also clearly induced during adaptation to detergent and heat, but not acid, stresses. The biological meaning of cysteine synthase in the context of adaptation to oxidative and non-oxidative stresses is discussed.     

Isolation of several anti-stress genes from the halotolerant green alga Chlamydomonas by simple functional expression screening with Escherichia coli

To isolate anti-stress genes from a halotolerant marine green alga, a simple screening method based on the bacterial expression system was examined. The method consisted of three steps: (i) directional cDNA library construction in a ZAPII vector, (ii) in vivo mass excision of a ZAPII library into phagemid DNA, and (iii) screening for anti-salt-stress and anti-oxidative-stress genes by culturing bacterial cells carrying the in vivo excised phagemid on selection agar plates with a high concentration of NaCl and/or methyl viologen (MV), and by isolating stress-tolerant bacterial colonies. By this method, we screened the cDNA library of halotolerant Chlamydomonas sp. strain W-80, and isolated several stress-related gene homologs, such as glutathione peroxidase, ascorbate peroxidase, bbc1 (breast basic conserved; a low temperature induced gene in Brassica napus), and cyanide-resistant alternative oxidase.     

Effect of beta-lactotensin on acute stress and fear memory

β-Lactotensin (β-LT) is a bioactive peptide derived from bovine milk β-lactoglobulin and is a natural ligand for neurotensin receptors. We examined the effect of β-LT on restraint stress and fear memory in mice. Mice subjected to acute restraint stress exhibited a decreased number of head-dips and increased head-dip latency compared to non-stressed controls in the hole-board test, reflecting increased stress-induced behaviors. However, prior administration of β-LT improved the behaviors caused by stress. The anti-stress effect of β-LT was blocked by levocabastine, a neurotensin receptor subtype 2 (NTR2) antagonist. In the fear-conditioning test, the duration of freezing responses by cued fear conditioning was significantly reduced in mice administered β-LT compared with control mice. These results suggest that β-LT has an anti-stress effect and promotes the extinction of fear memory, which may be mediated by NTR2.