The leukotriene B4 receptor BLT1 is stabilized by transmembrane helix capping mutations

In this study, we introduced structure-based rational mutations in the guinea pig leukotriene B₄ receptor (gpBLT1) in order to enhance the stabilization of the protein. Elements thought to be unfavorable for the stability of gpBLT1 were extracted based on the stabilization elements established in soluble proteins, determined crystal structures of G-protein-coupled receptors (GPCRs), and multiple sequence alignment. The two unfavorable residues His83^2.67 and Lys88^3.21, located at helix capping sites, were replaced with Gly (His83Gly^2.67 and Lys88Gly^3.21). The modified protein containing His83Gly^2.67/Lys88Gly^3.21 was highly expressed, solubilized, and purified and exhibited improved thermal stability by 4 °C in comparison with that of the original gpBLT1 construct. Owing to the double mutation, the expression level increased by 6-fold (B_max=311 pmol/mg) in the membrane fraction of Pichia pastoris. The ligand binding affinity was similar to that of the original gpBLT1 without the mutations. Similar unfavorable residues have been observed at helix capping sites in many other GPCRs; therefore, the replacement of such residues with more favorable residues will improve stabilization of the GPCR structure for the crystallization.     

A new matrix protein family related to the nacreous layer formation of Pinctada fucata

We have isolated a new matrix protein family (N16) which is specific to the nacreous layer of the Japanese pearl oyster, Pinctada fucata, and have cloned and characterized the cDNAs coding for the components. Analysis of the deduced amino acid sequence revealed that N16 showed no definitive homology with other proteins. The in vitro studies of the crystallization clarified that N16 induced aragonite crystals when fixed on the substrate but inhibited crystal formation without it. The aragonite crystals showed platy morphology different from those formed inorganically, and long intervals of incubation resulted in crystalline layers highly similar to the nacreous layer.     

Preresonance Raman studies of poly(L-lysine), poly(L-glutamic acid), and deuterated N-methylacetamides

The Raman spectra of poly(L -lysine) with various structures, ionized poly(L -glutamic acid), and deuterated N-methylacetamides have been observed using visible and the 257.3-nm laser lines as the light source. Most of the Raman bands with significantly enhanced intensities in the uv-excited spectra of the polymers have been assigned to the vibrations associated with the C?O and C–N stretching modes, the amide I, II, III, I′, II′, and III′, with reference to the results obtained for simple amide molecules including the deuterated N-methylacetamides. Several amide frequencies have been newly identified and the structures of the polymers have been discussed through the comparison of the Raman and ir amide frequencies.     

Abundance and population structure of ammonia-oxidizing bacteria that inhabit canal sediments receiving effluents from municipal wastewater treatment plants

A polyphasic, culture-independent study was conducted to investigate the abundance and population structure of ammonia-oxidizing bacteria (AOB) in canal sediments receiving wastewater discharge. The abundance of AOB ranged from 0.2 to 1.9% and 1.6 to 5.7% of the total bacterial fraction by real-time PCR and immunofluorescence staining, respectively. Clone analysis and restriction endonuclease analysis revealed that the AOB communities influenced by the wastewater discharge were dominated by Nitrosomonas, were similar to each other, and were less diverse than the communities outside of the immediate discharge zone.     

Evaluation of the mutagenic properties of the coccolithophorePleurochrysis carterae (Haptophyceae) as a potential human food supplement

The mutagenicity of the algaPleurochrysis carterae for use as human food was tested by the Ames method with the modification of pre-incubation, by usingSalmonella typhimurium TA98, TA100, TA1535, TA1537 andEscherichia coli WP2uvrA. The freeze-dried powder ofP. carterae was not mutagenic to any strain either with or without S9 mix. In view of the absence of adverse effects ofP. carterae in this mutagenicity study, it is suggested thatP. carterae is safe for human consumption as a human food supplement.Author for correspondence     

Studies on Bacterial Protease

A neutral protease of Bacillus subtilis var. amylosacchariticus was purified and crystallized by sequential chromatography on columns of Duolite A-2 anion-exchange resin, CM-cellulose and DEAE-sephadex A-50. The crystalline preparation was chromatographically homogeneous and confirmed to be monodispersive by physicochemical criteria. The enzyme was most active at near pH 7 against casein and stabilized by calcium salts. Some metalchelating agents and metal ions such as Hg?, Pb?, Cu? and Fe? markedly inactivated the enzyme, whereas diisopropyl phosphorofluoridate, sulfhydryl reagents and protease inhibitor of potato did not affect the activity. The neutral protease obtained here was rather stable as compared with the neutral protease ever reported and was able to be freeze-dried without any appreciable lose in enzyme activity.     

Oncogenic RAS-induced downregulation of ATG12 is required for survival of malignant intestinal epithelial cells

Activating mutations of RAS GTPase contribute to the progression of many cancers, including colorectal carcinoma. So far, attempts to develop treatments of mutant RAS-carrying cancers have been unsuccessful due to insufficient understanding of the salient mechanisms of RAS signaling. We found that RAS downregulates the protein ATG12 in colon cancer cells. ATG12 is a mediator of autophagy, a process of degradation and reutilization of cellular components. In addition, ATG12 can kill cells via autophagy-independent mechanisms. We established that RAS reduces ATG12 levels in cancer cells by accelerating its proteasomal degradation. We further observed that RAS-dependent ATG12 loss in these cells is mediated by protein kinases MAP2K/MEK and MAPK1/ERK2-MAPK3/ERK1, known effectors of RAS. We also demonstrated that the reversal of the effect of RAS on ATG12 achieved by the expression of exogenous ATG12 in cancer cells triggers both apoptotic and nonapoptotic signals and efficiently kills the cells. ATG12 is known to promote autophagy by forming covalent complexes with other autophagy mediators, such as ATG5. We found that the ability of ATG12 to kill oncogenic RAS-carrying malignant cells does not require covalent binding of ATG12 to other proteins. In summary, we have identified a novel mechanism by which oncogenic RAS promotes survival of malignant intestinal epithelial cells. This mechanism is driven by RAS-dependent loss of ATG12 in these cells.