Purification and Characteristics of an Autolytic Chitinase of Piromyces communis OTS1 from Culture Medium

An autolysis chitinase was purified from the cultural medium of the anaerobic fungus Piromyces communis OTS1 by ammonium sulfate precipitation, affinity chromatography with regenerated chitin, chromato-focusing, gel filtration, and chromato-focusing again. The optimal pH and temperature were 6.0 and 50°C, respectively, for a 20-min assay. The chitinase was stable from pH 6.0 to 8.0, but was unstable at 70°C for 20 min. The molecular mass of chitinase was estimated by SDS-PAGE to be 44.9 kDa, and its pI was 4.4. The enzyme activity, which was of the ‘endo’ type, was inhibited by Hg²⁺ and allosamidin. The chitinase hydrolyzes chitin powder and fungal cell walls at a higher rate than an artificial chitin substrate. It can be concluded that extracellular chitinase is similar to cytosolic chitinase, but they are not the same protein. Received: 3 December 1996 / Accepted: 28 January 1997     

Genes,enzymes and membrane proteins of the nitrate respiration system ofEscherichia coli

Summary A method was devised to isolate mutants carrying deletions through several genetic loci (chlD ⁺ andchlA ⁺) which are involved in the membrane-bound nitrate respiratory complex ofEscherichia coli. Specific transducing phages were used to reintroduce these genes. Comparisons of membrane fractions from these transduced strains showed five membrane proteins that are necessary for the formation of an active nitrate respiration system. Two particular bacterial genes (chlD ⁺ andchlA ⁺) were shown to control these five membrane proteins.Three of the proteins specified bychlA ⁺, appear to be constitutively controlled and always present in the membrane ofE. coli irrespective of growth conditions, while the other two proteins, specified bychlD ⁺, appear to be induced byanaerobic growth in the presence of nitrate.     

Persistent oxidative stress in human colorectal carcinoma, but not in adenoma.

Few studies have been conducted focusing on a potential role of reactive oxygen species in tumor cell metabolism. Here we studied human colorectal adenocarcinomas and adenomas to determine whether oxidative stress is imposed on cancer cells in vivo and used specific antibodies against 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE)-modified proteins, and 3-nitro-L-tyrosine (3-NT) to determine whether there is an association between oxidative stress and cellular proliferation. Higher levels of oxidative modifications in DNA and proteins were observed in carcinoma cells, but not in adenoma cells, than in the corresponding nontumorous epithelial cells by immunohistochemistry as well as high-performance liquid chromatography (HPLC)-based 8-OHdG determination. The fraction of proliferating cell nuclear antigen-positive cells was proportionally associated in adenocarcinomas with the staining intensities of 8-OHdG and 3-NT. Furthermore, Western blot analysis of the proteins extracted from carcinoma cells revealed several specific proteins modified by HNE or peroxynitrite. Thus we concluded that colorectal carcinoma, but not adenoma cells, are exposed to more oxidative stress than their corresponding nontumorous epithelial cells, regardless of clinical stage and histology, and further that the oxidative stress in carcinoma cells might stimulate cellular proliferation.     

Silibinin ameliorates Aβ<Subscript>25-35</Subscript>-induced memory deficits in rats by modulating autophagy and attenuating neuroinflammation as well as oxidative stress

Alzheimer’s disease (AD) is a progressive, neurodegenerative disease. Accumulating evidence suggests that inflammatory response, oxidative stress and autophagy are involved in amyloid β (Aβ)-induced memory deficits. Silibinin (silybin), a flavonoid derived from the herb milk thistle, is well known for its hepatoprotective activities. In this study, we investigated the neuroprotective effect of silibinin on Aβ_25-35-injected rats. Results demonstrated that silibinin significantly attenuated Aβ_25-35-induced memory deficits in Morris water maze and novel object-recognition tests. Silibinin exerted anxiolytic effect in Aβ_25-35-injected rats as determined in elevated plus maze test. Silibinin attenuated the inflammatory responses, increased glutathione (GSH) levels and decreased malondialdehyde (MDA) levels, and upregulated autophagy levels in the Aβ_25-35-injected rats. In conclusion, silibinin is a potential candidate for AD treatment because of its anti-inflammatory, antioxidant and autophagy regulating activities.