Overexpression of amyloid precursor protein alters its normal processing and is associated with neurotoxicity.

The recent discovery that point mutations in the beta/A4 amyloid precursor protein may be the cause of certain forms of familial Alzheimer's disease provides strong support for the view that a thorough understanding of the metabolism of this protein may elucidate the pathogenesis of most forms of the disease and thus serve as a basis for rational prevention and therapy. Here we show that overexpression of a portion of the amyloid precursor protein molecule produces at least four distinct fragments of the COOH-terminus of amyloid precursor protein, suggesting altered proteolysis of amyloid precursor protein, and that such overexpression is associated with cytotoxicity. The degree of toxicity in the P19 cell culture model (differentiating mouse embryonal carcinoma cells) is shown to be related to the two larger novel COOH-terminal protein fragments (16 and 14 kilodalton), as well as to levels of expression of these two fragments. The toxicity is manifested in several differentiated cell lineages, including neuronal cells.     

Viridiuvalis adhaerens gen. et sp. nov., a novel colony-forming chlorarachniophyte

Journal of Plant Research - A new chlorarachniophyte, Viridiuvalis adhaerens gen. et sp. nov. was isolated from the mucus on a coral reef from Zanpa Beach, Okinawa, Japan. The main vegetative stage...     

Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation

Smad7 is an inhibitory Smad that acts as a negative regulator of signaling by the transforming growth factor-beta (TGF-beta) superfamily proteins. Smad7 is induced by TGF-beta, stably interacts with activated TGF-beta type I receptor (TbetaR-I), and interferes with the phosphorylation of receptor-regulated Smads. Here we show that Smurf1, an E3 ubiquitin ligase for bone morphogenetic protein-specific Smads, also interacts with Smad7 and induces Smad7 ubiquitination and translocation into the cytoplasm. In addition, Smurf1 associates with TbetaR-I via Smad7, with subsequent enhancement of turnover of TbetaR-I and Smad7. These results thus reveal a novel function of Smad7, i.e. induction of degradation of TbetaR-I through recruitment of an E3 ligase to the receptor.     

Identification of a highly immunogenic mouse breast cancer sub cell line, 4T1-S

Cancer vaccines serve as a promising clinical immunotherapeutic strategy that help to trigger an effective and specific antitumor immune response compared to conventional therapies. However, poor immunogenicity of tumor cells remains a major obstacle for clinical application, and developing new methods to modify the immunogenicity of tumor cells may help to improve the clinical outcome of cancer vaccines. 4T1 mouse breast cancer cell line has been known as poorly immunogenic and highly metastatic cell line. Using this model, we identified a sub cell line of 4T1—designated as 4T1-Sapporo (4T1-S)—which shows immunogenic properties when used as a vaccine against the same line. In 4T1-S-vaccinated mice, subcutaneous injection of 4T1-S resulted in an antitumor inflammatory response represented by significant enlargement of draining lymph nodes, accompanied with increased frequencies of activated CD8 T cells and a subpopulation of myeloid cells. Additionally, 4T1-S vaccine was ineffective to induce tumor rejection in nude mice, which importantly indicate that 4T1-S vaccine rely on T cell response to induce tumor rejection. Further analysis to identify mechanisms that control tumor immunogenicity in this model may help to develop new methods for improving the efficacies of clinical cancer vaccines.     

Regulation of L-alanine-initiated germination ofBacillus subtilis spores by alanine racemase

Summary Germination ofBacillus subtilis spores was initiated by L-Ala and competitively inhibited by D-Ala, suggesting the presence of an alanine receptor. The spores showed alanine racemase activity in the spore coat. To investigate the role of alanine racemase (L D) on germination, net racemase activity was determined using diphenylamine as a germination inhibitor and germination was measured using D-penicillamine as a racemase inhibitor. Apparent affinity of L-Ala to the germinant receptor was more than 1000 times higher than that to the racemase. Germination increased in the presence of D-penicillamine, when the concentration of L-Ala was low and that of spores was high. Racemase activity was optimal at 65°C at pH 9.0 and germination at 43°C at pH 7.2. Under unfavorable growth conditions such as high population of spores in limited nutrients, high temperature and high pH, spore alanine racemase converted the germinant actively to the inhibitor and this conversion may regulate germination for survival of the population.     

Purification and some properties of a soluble cytochrome (cytochrome c-551) from Erythrobacter species strain OCh 114

A soluble cytochrome, cytochrome c-551 was purified from an aerobic photosynthetic bacterium Erythrobacter species strain OCh 114 (ATCC No. 33942) by ammonium sulfate fractionation, ion-exchange chromatography and gel-filtration. The cytochrome had absorption maxima at 277, 410, and 524–525 nm in the oxidized form, and at 415, 522, and 550.5 nm in the reduced form. At 77 K, the -band of the absorption spectrum of the reduced form split in two at 547 and 549 nm. The millimolar absorption coefficient at 550.5 nm was 26.8 mM^-1 cm^-1 in the reduced form. This cytochrome was an acidic protein with an isoelectric point of 4.9. Its molecular weight was determined to be 15,000 by gel-filtration on Sephadex G-100 and 14,500 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The midpoint potential of this cytochrome was +250 mV at pH 7.0. This cytochrome did not bind CO.     

Cysteine-to-serine shuffling using a Saccharomyces cerevisiae expression system improves protein secretion: case of a nonglycosylated mutant of miraculin, a taste-modifying protein

PURPOSE OF WORK: Soluble protein expression is an important first step during various types of protein studies. Here, we present the screening strategy of secretable mutant. The strategy aimed to identify those cysteine residues that provoke protein misfolding in the heterologous expression system. Intentional mutagenesis studies should consider the size of the library and the time required for expression screening. Here, we proposed a cysteine-to-serine shuffling mutation strategy (CS shuffling) using a Saccharomyces cerevisiae expression system. This strategy of site-directed shuffling mutagenesis of cysteine-to-serine residues aims to identify the cysteine residues that cause protein misfolding in heterologous expression. In the case of a nonglycosylated mutant of the taste-modifying protein miraculin (MCL), which was used here as a model protein, 25% of all constructs obtained from CS shuffling expressed MCL mutant, and serine mutations were found at Cys47 or Cys92, which are involved in the formation of the disulfide bond. This indicates that these residues had the potential to provoke protein misfolding via incorrect disulfide bonding. The CS shuffling can be performed using a small library and within one week, and is an effective screening strategy of soluble protein expression.