Metabolism of triacylglycerol in Mycobacterium smegmatis.

Mycobacterium smegmatis cells incorporated [1-14C]oleic acid into triacylglycerols (TG) from the medium more rapidly than shorter chain fatty acids, caprilic and butyric acids. This incorporation was inhibited more strongly by 10(-3) M N-ethylmaleimide than by 10(-3) M KCN. [14C]TG in the bacterial cells was utilized when the cells were in poor nutritional conditions, such as phosphate buffer (pH 7.0) containing oleic acid. Accumulation of TG was observed in the cells at late stages of growth. Diglyceride acyltransferase [EC 2.3.1.20] activity was detected in a cell-free extract from this bacterium. The pH optimum of this enzyme was between pH 7 and 9. F- and Tween 20 showed remarkable enhancing and inhibitory effects, respectively.     

Recessive and dominant genes controlling inducible sexual agglutinability in Saccharomyces cerevisiae

Summary We have characterized the two dominant genes, IND 1 and IND 2, responsible for inducible sexual agglutinability. The strains carrying these genes differ from the inducible strains carrying the recessive gene, saa 1 in the following points. The former strains produce agglutination substance at 22°, 28°, and 37° C only in response to sex pheromone of the opposite mating type, but the latter strains produce the substance constitutively without the pheromone at 22° C, only in response to the pheromone at 28° C, and do not produce the substance, even in the presence of the pheromone, at 37° C.We suggest that strains carrying one of the dominant, inducible genes are wild type and have a pheromone-controlled regulatory system of sexual agglutinability.     

Selective degradation of altered tomato β-fructofuranosidase molecules by neutral protease

Evidence is presented for the selective breakdown of altered tomato β-fructofuranosidase molecules by a neutral protease from Bacillus subtilis.     

Sulfonamide-based non-alkyne LpxC inhibitors as Gram-negative antibacterial agents

We attempted to optimize sulfonamide-based non-alkyne LpxC inhibitors by focusing on improvements in enzyme inhibitory and antibacterial activity. It was discovered that inhibitors possessing 2-aryl benzofuran as a hydrophobe exhibited good activity. In particular, compound 21 displayed impressive antibacterial activity (E. coli MIC = 0.063 μg/mL, K. pneumoniae MIC = 0.5 μg/mL, and P. aeruginosa MIC = 0.5 μg/mL), and is a promising lead for further exploration as an antibacterial agent.     

miR-33a-5p in small extracellular vesicles as non-invasive biomarker for oxaliplatin sensitivity in human colorectal cancer cells

microRNAs (miRNAs) contained in small extracellular vesicles (sEVs) are candidates for non-invasive biomarkers. Oxaliplatin (L-OHP) has been approved for advanced colorectal cancer (CRC) chemotherapy. However, the response to L-OHP differs among CRC patients. In addition, CRC cells often acquire the resistance to L-OHP. This study aimed at the prediction of L-OHP sensitivity by measuring extracellular miRNAs levels. Firstly, we compared intracellular miRNAs expressions in L-OHP-sensitive CRC cells (SW620 and HCT116 cells) with those in acquired and intrinsic L-OHP-resistant cells. In microarray and real-time RT-PCR analyses, the intracellular miR-33a-5p, miR-210–3p, and miR-224–5p expressions were lower in acquired and intrinsic L-OHP-resistant CRC cells than sensitive cells. Furthermore, in SW620 cells, L-OHP sensitivity was decreased by miR-33a-5p inhibitor. On the other hand, miR-210–3p or miR-224–5p inhibitor did not affect L-OHP sensitivity in SW620 cells. Secondly, the amount of miR-33a-5p, miR-210–3p, and miR-224–5p in sEVs was compared. The amount of miR-33a-5p and miR-210–3p in sEVs secreted from acquired and intrinsic L-OHP-resistant cells tended to be small. miR-224–5p was not detected in sEVs secreted from three types of CRC cells examined. To the best of our knowledge, this is the first study demonstrating that miR-33a-5p and/or miR-210–3p in sEVs would be candidates for biomarkers of L-OHP sensitivity. In particular, miR-33a-5p is a promising candidate because it would be directly involved in L-OHP sensitivity.     

α5β1 integrin induces the expression of noncartilaginous procollagen gene expression in articular chondrocytes cultured in monolayers

Introduction

Articular chondrocytes undergo an obvious phenotypic change when cultured in monolayers. During this change, or dedifferentiation, the expression of type I and type III procollagen is induced where normal chondrocytes express little type I and type III procollagen. In this study, we attempted to determine the mechanism(s) for the induction of such procollagen expression in dedifferentiating chondrocytes.

Methods

All experiments were performed using primary-cultured human articular chondrocytes under approval of institutional review boards. Integrin(s) responsible for the induction of type I and type III procollagen expression were specified by RNAi experiments. The signal pathway(s) involved in the induction were determined by specific inhibitors and RNAi experiments. Adenovirus-mediated experiments were performed to identify a small GTPase regulating the activity of integrins in dedifferentiating chondrocytes. The effect of inhibition of integrins on dedifferentiation was investigated by experiments using echistatin, a potent disintegrin. The effect of echistatin was investigated first with monolayer-cultured chondrocytes, and then with pellet-cultured chondrocytes.

Results

In dedifferentiating chondrocytes, α5β1 integrin was found to be involved in the induction of type I and type III procollagen expression. The induction was known to be mediated by v-akt murine thymoma viral oncogene homolog (AKT) signaling. Among the three AKT isoforms, AKT1 seemed to be most involved in the signaling. Elated RAS viral (r-ras) oncogene homolog (RRAS) was considered to regulate the progression of dedifferentiation by modulating the affinity and avidity of α5β1 integrin to ligands. Echistatin inhibited dedifferentiation of monolayer-cultured chondrocytes. Furthermore, the matrix formed by pellet-cultured chondrocytes more closely resembled that of normal cartilage compared with the controls.

Conclusions

The result of this study has shown, for the first time, that α5β1 integrin may be responsible for the induction of non-cartilaginous collagen expression in chondrocytes undergoing dedifferentiation. Again, this study has shown that the inhibition of ligand ligation to integrins may be an effective strategy to inhibit phenotypic change of cultured chondrocytes, and to improve the quality of matrix synthesized by primary cultured chondrocytes.     

The role of disulfide bond in hyperthermophilic endocellulase

The hyperthermophilic endocellulase, EGPh (glycosyl hydrolase family 5) from Pyrococcus horikoshii possesses 4 cysteine residues forming 2 disulfide bonds, as identified by structural analysis. One of the disulfide bonds is located at the proximal region of the active site in EGPh, which exhibits a distinct pattern from that of the thermophilic endocellulase EGAc (glycosyl hydrolase family 5) of Acidothermus cellulolyticus despite the structural similarity between the two endocellulases. The structural similarity between EGPh and EGAc suggests that EGPh possesses a structure suitable for changing the position of the disulfide bond corresponding to that in EGAc. Introduction of this alternative disulfide bond in EGPh, while removing the original disulfide bond, did not result in a loss of enzymatic activity but the EGPh was no longer hyperthermostable. These results suggest that the contribution of disulfide bond to hyperthermostability at temperature higher than 100 °C is restrictive, and that its impact is dependent on the specific structural environment of the hyperthermophilic proteins. The data suggest that the structural position and environment of the disulfide bond has a greater effect on high-temperature thermostability of the enzyme than on the potential energy of the dihedral angle that contributes to disulfide bond cleavage.