Characterization of a proteolytic enzyme derived from a Bacillus strain that effectively degrades prion protein

AIMS: The purpose of this paper was to screen candidate bacterial strains for the production of proteases suitable for application to the degradation of pathogenic forms of prion protein (PrP(Sc)). This paper describes the biochemical characteristics and proteolytic activity of the isolated protease. METHODS AND RESULTS: After screening more than 200 bacterial proteases for keratinolytic activity, we identified a Bacillus stain that produced a protease exhibiting high-degradation activity against a scrapie PrP(Sc). Sequence analysis indicated that this serine-protease belonged to the Subtilisin family and had optimum pH and temperature ranges of 9-10 and 60-70 degrees C. Western blotting analysis revealed that the protease was also capable of decomposing bovine spongiform encephalopathy-infected brain homogenate. In addition, the protease was demonstrated to degrade dried PrP(Sc) that had become firmly attached to a plastic surface considerably more effectively than proteinase K or PWD-1, a previously reported keratinase. CONCLUSIONS: These results indicate that the isolated protease exhibited higher activity for PrP(Sc) degradation compared with other proteases examined. SIGNIFICANCE AND IMPACT OF THE STUDY: This protease could be used under moderate conditions for the decontamination of precision instruments that are susceptible to PrP(Sc) contamination.     

In vitro and in vivo anti-inflammatory activity of digested peptides derived from salmon myofibrillar protein conjugated with a small quantity of alginate oligosaccharide

Salmon myofibrillar protein (Mf) was investigated as a source of edible anti-inflammatory products. Peptides produced by stepwise digestion of Mf (without carbohydrate) with pepsin and trypsin had little effect on the secretion of inflammation-related compounds from lipopolysaccharide-stimulated RAW 264.7 macrophage cells. However, peptides prepared from Mf conjugated with alginate oligosaccharide (AO; 19 μg/mg protein) (dMSA) through the Maillard reaction in the presence of sorbitol significantly reduced the secretion of the pro-inflammatory mediators nitric oxide, tumor necrosis factor (TNF)-α and interleukin (IL)-6, as well as mRNA expression of TNF-α, IL-6, inducible nitric oxide synthase and cyclooxygenase-2. Additionally, dMSA inhibited acute inflammation in a carrageenan-induced model of paw edema in mice, but had no effect on natural killer cell cytotoxic activity or macrophage phagocytosis. These results suggest that fish Mf conjugated with AO may be a potential food material with anti-inflammatory function.     

Biosynthetic pathway of phytosiderophores in iron-deficient graminaceous plants: A new assay system for the detection of nicotianamine amino-transferase activity

A new assay system for the detection of nicotianamine amino-transferase activity was developed. The activity of nicotianamine amino-transferase which participated in biosynthetic pathway of MAs from methionine in graminaceous plants was induced by the iron deficiency treatment.     

Serum stability of selected decapeptide agonists of KISS1R using pseudopeptides

Metastin/kisspeptin, a 54-amino acid peptide, is the ligand of the G-protein-coupled receptor KISS1R which plays a key role in pathways that regulate reproduction and cell migration in many endocrine and gonadal tissues. The N-terminally truncated decapeptide, metastin(45–54), has 3–10 times higher receptor affinity and intracellular calcium ion-mobilizing activity but is rapidly inactivated in serum. In this study we designed and synthesized stable KISS1R agonistic decapeptide analogs with selected substitutions at positions 47, 50, and 51. Replacement of glycine with azaglycine (azaGly) in which the α-carbon is replaced with a nitrogen atom at position 51 improved the stability of amide bonds between Phe⁵⁰-Gly⁵¹ and Gly⁵¹-Leu⁵² as determined by in vitro mouse serum stability studies. Substitution for tryptophan at position 47 with other amino acids such as serine, threonine, β-(3-pyridyl)alanine, and d-tryptophan (d-Trp), produced analogs that were highly stable in mouse serum. d-Trp⁴⁷ analog 13 showed not only high metabolic stability but also excellent KISS1R agonistic activity. Other labile peptides may have increased serum stability using amino acid substitution.     

Induced activity of adenine phosphoribosyltransferase (APRT) in iron-deficiency barley roots: a possible role for phytosiderophore production

To isolate the genes involved in the response of graminaceous plants to Fe-deficient stress, a protein induced by Fe-deficiency treatment was isolated from barley (Hordeum vulgare L.) roots. Based on the partial amino acid sequence of this protein, a cDNA (HvAPT1) encoding adenine phosphoribosyltransferase (APRT: EC 2.4.2.7) was cloned from a cDNA library prepared from Fe-deficient barley roots. Southern analysis suggested that there were at least two genes encoding APRT in barley. Fe deficiency increased HvAPT1 expression in barley roots and resupplying Fe to the Fe-deficient plants rapidly negated the increase in HvAPT1 mRNA. Analysis of localization of HvAPT1-sGFP fusion proteins in tobacco BY-2 cells indicated that the protein from HvAPT1 was localized in the cytoplasm of cells. Consistent with the results of Northern analysis, the enzymatic activity of APRT in barley roots was remarkably increased by Fe deficiency. This induction of APRT activity by Fe deficiency was also observed in roots of other graminaceous plants such as rye, maize, and rice. In contrast, the induction was not observed to occur in the roots of a non-graminaceous plant, tobacco. Graminaceous plants generally synthesize the mugineic acid family phytosiderophores (MAs) in roots under Fe-deficient conditions. In this paper, a possible role of HvAPT1 in the biosynthesis of MAs related to adenine salvage in the methionine cycle is discussed.     

Cloning of Nicotianamine Synthase Genes, Novel Genes Involved in the Biosynthesis of Phytosiderophores

Nicotianamine synthase (NAS), the key enzyme in the biosynthetic pathway for the mugineic acid family of phytosiderophores, catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine. We purified NAS protein and isolated the genes nas1, nas2, nas3, nas4, nas5-1, nas5-2, and nas6, which encode NAS and NAS-like proteins from Fe-deficient barley (Hordeum vulgare L. cv Ehimehadaka no. 1) roots. Escherichia coli expressing nas1 showed NAS activity, confirming that this gene encodes a functional NAS. Expression of nas genes as determined by northern-blot analysis was induced by Fe deficiency and was root specific. The NAS genes form a multigene family in the barley and rice genomes.     

Modulation by cAMP of 1alpha,25-dihydroxyvitamin D3 sensitivity of murine erythroleukemia cells.

As we previously reported, 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently inhibited not only proliferation of undifferentiated murine erythroleukemia (MEL) cells but also activin A-induced erythroid differentiation of MEL cells. However, the effect of 1,25(OH)2D3 on MEL cell proliferation was significantly greater by one order of magnitude than that on differentiation (IC(50): 9.2 vs 0.8 nM, respectively). The response of activin A-treated mature MEL cells to 1,25(OH)2D3 in the induction of 1,25(OH)2D3-24-hydroxylase (24-OHase) activity, a rapid effect of 1,25(OH)2D3, was enhanced to the same degree as in untreated immature cells, suggesting that differences in capacity of cells to inactivate 1,25(OH)2D3 did not contribute to augmentation of 1,25(OH)2D3 effect in activin A-treated mature cells. Furthermore, neither the number nor the affinity of vitamin D receptors (VDR) differed significantly between activin A-treated cells and untreated immature cells. The intracellular cAMP level, which affects 1,25(OH)2D3-mediated induction of 24-OHase activity, was significantly less in activin A-treated mature cells than in immature MEL cells. The addition of dibutyryl cAMP (dbc AMP) to activin A-treated MEL cells dose-dependently attenuated 1,25(OH)2D3-mediated induction of 24-OHase activity, finally to a level comparable to that of the untreated cells at the final concentration of 100 nM dbcAMP, while dbcAMP itself by 100 nM did not affect MEL cell differentiation by 24 h. In summary, we have shown for the first time that 1,25(OH)2D3 exerted its effect on leukemia cells at physiological concentration and that the magnitude of this effect depended on the changes in intracellular cAMP level through stages of differentiation, suggesting that the cAMP-protein kinase A system may be useful as a target for clinical application of vitamin D analogs by improving the sensitivity of leukemic cells to 1,25(OH)2D3.     

Transformation of chicken embryo fibroblast cells by avian retroviruses containing the human Fyn gene and its mutated genes.

The transforming activity of the human fyn protein, p59fyn, which is a kinase of the src family, was investigated by testing the effect of recombinant avian retrovirus (Fyn virus) expressing p59fyn on chickens or cultured chicken embryo fibroblast (CEF) cells. The Fyn virus did not induce transformed foci. After several passages of the virus stock on CEF cells, however, a few foci were detected in the presence of dimethyl sulfoxide. Chickens inoculated with Fyn virus at the stage of 12-day-old embryos developed fibrosarcomas 3 to 6 weeks after hatching. The viruses obtained from these foci and from one of the tumor tissues showed high transforming activity in the presence of dimethyl sulfoxide, suggesting that these viruses carry spontaneous mutations of the fyn gene. Four fyn genes from CEF DNAs infected with transforming viruses were molecularly cloned, and their products were confirmed to possess transforming activity. DNA sequence analysis of the fyn genes showed that two of the four mutants have Thr instead of Ile at position 338 in the kinase domain. The other two mutants carry deletions of 78 and 108 base pairs, respectively, which result in complete loss of region C of SH2. The overall level of proteins containing phosphotyrosine was significantly higher in transformed cells than in normal CEF cells. Our data indicate that when expressed at high levels in a retrovirus, normal p59fyn cannot cause cellular transformation, but that mutant p59fyn with either a single amino acid substitution in the kinase domain or a deletion including region C produces a transforming protein, perhaps due to enhanced tyrosine kinase activity. This is the first observation that deletion of region C can unmask the potential transforming activity of a src family kinase.