The canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner

To better understand the role of the canonical Wnt signaling pathway in cartilage development, we adenovirally expressed a constitutively active (ca) or a dominant negative (dn) form of lymphoid enhancer factor-1 (LEF-1), the main nuclear effector of the pathway, in undifferentiated mesenchymal cells, chondrogenic cells, and primary chondrocytes, and examined the expression of markers for chondrogenic differentiation and hypertrophy. caLEF-1 and LiCl, an activator of the canonical pathway, promoted both chondrogenic differentiation and hypertrophy, whereas dnLEF-1 and the gene silencing of beta-catenin suppressed LiCl-promoted effects. To investigate whether these effects were dependent on Sox9, a master regulator of cartilage development, we stimulated Sox9-deficient ES cells with the pathway. caLEF-1 and LiCl promoted both chondrogenic differentiation and hypertrophy in wild-type, but not in Sox9-deficient, cells. The response of Sox9-deficient cells was restored by the adenoviral expression of Sox9. Thus, the canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner.     

Involvement of endogenous bone morphogenetic protein (BMP) 2 and BMP6 in bone formation

Although accumulated evidence has shown the bone anabolic effects of bone morphogenetic proteins (BMPs) that were exogenously applied in vitro and in vivo, the roles of endogenous BMPs during bone formation remain to be clarified. This study initially investigated expression patterns of BMPs in the mouse long bone and found that BMP2 and BMP6 were the main subtypes expressed in hypertrophic chondrocytes that induce endochondral bone formation. We then examined the involvement of the combination of these BMPs in bone formation in vivo by generating the compound-deficient mice (Bmp2+/-;Bmp6-/-). Under physiological conditions, these mice exhibited moderate growth retardation compared with the wild-type (WT) littermates during the observation period up to 52 weeks of age. Both the fetal and adult compound-deficient mice showed a reduction in the trabecular bone volume with suppressed bone formation, but normal bone resorption, whereas the single deficient mice (Bmp2+/- or Bmp6-/-) did not. When a fracture was created at the femoral midshaft and the bone healing was analyzed, the endochondral bone formation, but not intramembranous bone formation, was impaired by the compound deficiency. In the cultures of bone marrow cells, however, there was no difference in osteogenic differentiation between WT and compound-deficient cells in the presence or absence of the exogenous BMP2. We thus concluded that endogenous BMP2 and BMP6 cooperatively play pivotal roles in bone formation under both physiological and pathological conditions.     

Thermal stability of Rhizopus niveus lipase expressed in a kex2 mutant yeast

Lipase from Rhizopus niveus (RNL) has a complex structure, and recombinant RNL, has even more complex structural properties in the yeast, Saccharomyces cerevisiae. These properties are due to the processing and to the size of the glycosylated sugar chain. The processing site was presumed to be that for the proteinase product of the KEX2 gene in yeast. We therefore, constructed an expression system in which the KEX2 gene was disrupted to produce a non-processed type of lipase with high thermal stability. This type of lipase was thermally stable to a temperature 15 degrees C higher than that of each processed type of lipase. This non-processed lipase had 50% residual activity after 2 h at 50 degrees C, while the residual activity of the processed lipases was only 10% after 30-45 min of incubation at 50 degrees C. The CD spectrum of the non-processed type of lipase at 222 nm was almost unchanged by heating, suggesting that this group of lipases had a very rigid structure and that the peptide bond between the A- and B-chain contributed to maintain this rigid structure. On the other hand, the length of the sugar chain bound to the lipase had no effect on the thermal stability.     

A novel osteogenic helioxanthin-derivative acts in a BMP-dependent manner

To effectively treat serious bone defects using bone regenerative medicine, there is a need for the development of a small chemical compound that potently induces bone formation. We now report a novel osteogenic helioxanthin-derivative, TH. TH induced osteogenic differentiation in MC3T3-E1 cells, mouse primary osteoblasts, and mouse embryonic stem cells. The combination of TH and bone morphogenetic protein (BMP) 2 induced the mRNA expression of osteoblast marker genes and calcification in primary fibroblasts. The TH induced the mRNA of the inhibitor of DNA-binding 1 (Id-1), and its osteogenic effect was inhibited by Smad6 or Noggin. Furthermore, TH induced the mRNA expression of Bmp4 and Bmp6. These data suggest that TH exerts its potent osteogenic effect in a BMP-dependent manner by enhancing the effects of the existing BMPs and/or increasing the expression of Bmp4 and Bmp6. TH may help establish a more efficient bone regeneration system.     

Determination of 3-hydroxyisovalerylcarnitine and other acylcarnitine levels using liquid chromatography-tandem mass spectrometry in serum and urine of a patient with multiple carboxylase deficiency

Due to its increased concentration in blood, 3-hydroxyisovalerylcarnitine (C5OH-I) is an important indicator for the diagnosis of organic acidemias in newborns. However, C5OH-I has not been used as a standard in tandem mass spectrometric (MS/MS) assays because its isolation is difficult. We developed a new synthesis of C5OH-I and investigated its behavior by MS/MS. A method using the multiple reaction monitoring (MRM) mode of MS/MS with HPLC was developed which provides high accuracy, precision and reproducibility. Acylcarnitine profiles in the serum and urine of a patient with multiple carboxylase deficiency (MCD) showed increased levels compared to a healthy patient.     

A luminol chemiluminescence method for sensing histidine and lysine using enzyme reactions

The analysis of free amino acids in urine and plasma is useful for estimating disease status in clinical diagnoses. Changes in the concentration of free amino acids in foods are also useful markers of freshness, nutrition, and taste. In this study, the specific interaction between aminoacyl–tRNA synthetase (aaRS) and its corresponding amino acid was used to measure amino acid concentrations. Pyrophosphate released by the amino acid–aaRS binding reaction was detected by luminol chemiluminescence; the method provided selective quantitation of 1.0–30 μM histidine and 1.0–60 μM lysine.     

The c‐MYC‐ABCB5 axis plays a pivotal role in 5‐fluorouracil resistance in human colon cancer cells

c‐MYC overexpression is frequently observed in various cancers including colon cancer and regulates many biological activities such as aberrant cell proliferation, apoptosis, genomic instability, immortalization and drug resistance. However, the mechanism by which c‐MYC confers drug resistance remains to be fully elucidated. In this study, we found that the c‐MYC expression level in primary colorectal cancer tissues correlated with the recurrence rate following 5‐fluorouracil (5‐FU)‐based adjuvant chemotherapy. Supporting this finding, overexpression of exogenous c‐MYC increased the survival rate following 5‐FU treatment in human colon cancer cells, and knockdown of endogenous c‐MYC decreased it. Furthermore, c‐MYC knockdown decreased the expression level of ABCB5, which is involved in 5‐FU resistance. Using a chromatin immunoprecipitation assay, we found that c‐MYC bound to the ABCB5 promoter region. c‐MYC inhibitor (10058‐F4) treatment inhibited c‐MYC binding to the ABCB5 promoter, leading to a decrease in ABCB5 expression level. ABCB5 knockdown decreased the survival rate following 5‐FU treatment as expected, and the ABCB5 expression level was increased in 5‐FU‐resistant human colon cancer cells. Finally, using a human colon cancer xenograft murine model, we found that the combined 5‐FU and 10058‐F4 treatment significantly decreased tumorigenicity in nude mice compared with 5‐FU or 10058‐F4 treatment alone. 10058‐F4 treatment decreased the ABCB5 expression level in the presence or absence of 5‐FU. In contrast, 5‐FU treatment alone increased the ABCB5 expression level. Taken together, these results suggest that c‐MYC confers resistance to 5‐FU through regulating ABCB5 expression in human colon cancer cells.     

Polymerization of L- and DL-phenylalanine N-carboxyanhydride by poly(N-methyl-L-alanine)

Polymerizations of L - and DL -phenylalanine N-carboxyanhydride in nitrobenzene by poly (N-methyl-L -alanine) of varying degrees of polymerization (n = 1–30) were investigated. Poly(N-methyl-L -alanine) was prepared by the polymerization of N-methyl-L -alanine NCA with N-methyl-L -alanine diethylamide and the degree of polymerization was controlled by the molar ratio [NCA]/[Catalyst] + 1. This polymer was shown to be an asymmetrically selective catalyst which polymerized L -phenylalanine NCA at a faster rate than DL -phenylalanine NCA. With increasing degree of polymerization the stability of the secondary structure of poly(N-methyl-L -alanine) increased. This was confirmed by circular dichroism spectra. However, the degree of asymmetric selection did not increase as the stability of the secondary structure of poly(N-methyl-L -alanine) increased. These findings indicate that the interaction of a growing polypeptide in an ordered structure with NCA molecules prior to the reaction does not lead to an asymmetric selection, and that the mechanism of the asymmetric selection by poly(N-methyl-L -alanine) should be different from those proposed so far.     

Amino acid sensing using aminoacyl-tRNA synthetase

The detection of amino acids using aminoacyl-tRNA synthetases (ARSs) as the molecular recognition element was proposed, and the binding activity and specificity of ARSs were evaluated. Using this rapid and easy method, from 15 to 50 μM tyrosine could be measured specifically. The method suggested in this article could be realized without an amino acid labeling process or a large volume of organic solvents, and the time for measurement was reasonable.