Accumulation of oxidative stress around the stroke-like lesions of MELAS patients

To investigate the relationship between oxidative stress and progressive spread of the stroke-like lesions in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) with 3243A>G mutation, we retrospectively analyzed the spread frequency in patients with and without treatment with the radical scavenger edaravone. Oxidative damage and defensive enzymes were histologically evaluated. Spread was significantly less frequent in the patients treated with edaravone. Although 8-hydroxy-2′-deoxyguanosine, a marker for oxidative damage of DNA, was obviously accumulated in peri-lesional surviving neurons, manganese superoxide dismutase and 8-oxoguanine glycosylase 1 were not up-regulated in those neurons. Increased oxidative stress and insufficient defense could be involved in the pathogenesis of the spreading lesions in MELAS.     

Crystal structure of the galectin-9 N-terminal carbohydrate recognition domain from Mus musculus reveals the basic mechanism of carbohydrate recognition

The galectins are a family of beta-galactoside-binding animal lectins with a conserved carbohydrate recognition domain (CRD). They have a high affinity for small beta-galactosides, but binding specificity for complex glycoconjugates varies considerably within the family. The ligand recognition is essential for their proper function, and the structures of several galectins have suggested their mechanism of carbohydrate binding. Galectin-9 has two tandem CRDs with a short linker, and we report the crystal structures of mouse galectin-9 N-terminal CRD (NCRD) in the absence and the presence of four ligand complexes. All structures form the same dimer, which is quite different from the canonical 2-fold symmetric dimer seen for galectin-1 and -2. The beta-galactoside recognition mechanism in the galectin-9 NCRD is highly conserved among other galectins. In the apo form structure, water molecules mimic the ligand hydrogen-bond network. The galectin-9 NCRD can bind both N-acetyllactosamine (Galbeta1-4GlcNAc) and T-antigen (Galbeta1-3GalNAc) with the proper location of Arg-64. Moreover, the structure of the N-acetyllactosamine dimer (Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc) complex shows a unique binding mode of galectin-9. Finally, surface plasmon resonance assay showed that the galectin-9 NCRD forms a homophilic dimer not only in the crystal but also in solution.     

Circadian disruption accelerates tumor growth and angio/stromagenesis through a Wnt signaling pathway

Epidemiologic studies show a high incidence of cancer in shift workers, suggesting a possible relationship between circadian rhythms and tumorigenesis. However, the precise molecular mechanism played by circadian rhythms in tumor progression is not known. To identify the possible mechanisms underlying tumor progression related to circadian rhythms, we set up nude mouse xenograft models. HeLa cells were injected in nude mice and nude mice were moved to two different cases, one case is exposed to a 24-hour light cycle (L/L), the other is a more "normal" 12-hour light/dark cycle (L/D). We found a significant increase in tumor volume in the L/L group compared with the L/D group. In addition, tumor microvessels and stroma were strongly increased in L/L mice. Although there was a hypervascularization in L/L tumors, there was no associated increase in the production of vascular endothelial cell growth factor (VEGF). DNA microarray analysis showed enhanced expression of WNT10A, and our subsequent study revealed that WNT10A stimulates the growth of both microvascular endothelial cells and fibroblasts in tumors from light-stressed mice, along with marked increases in angio/stromagenesis. Only the tumor stroma stained positive for WNT10A and WNT10A is also highly expressed in keloid dermal fibroblasts but not in normal dermal fibroblasts indicated that WNT10A may be a novel angio/stromagenic growth factor. These findings suggest that circadian disruption induces the progression of malignant tumors via a Wnt signaling pathway.     

Anti-prion activity of protein-bound polysaccharide K in prion-infected cells and animals

Protein-bound polysaccharide K (PSK) is a clinical immunotherapeutic agent that exhibits various biological activities, including anti-tumor and anti-microbial effects. In the present study, we report on the anti-prion activity of PSK. It inhibited the formation of protease-resistant abnormal prion protein in prion-infected cells without any apparent alterations in either the normal prion protein turnover or the autophagic function in the cells. Its anti-prion activity was predominantly composed of the high molecular weight component(s) of the protein portion of PSK. A single subcutaneous dose of PSK slightly but significantly prolonged the survival time of peritoneally prion-infected mice, but PSK-treated mice produced neutralizing antibodies against the anti-prion activity of PSK. These findings suggest that PSK is a new anti-prion substance that may be useful in elucidating the mechanism of prion replication, although the structure of the anti-prion component(s) of PSK requires further evaluation.     

Efficient expansion of mouse primary tenocytes using a novel collagen gel culture method

Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell–cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation.