A comparison of the properties and the solution structure for RNA and DNA quadruplexes which contain two GGAGG sequences joined with a tetranucleotide linker

We have determined solution structure of r(GGAGGUUUUGGAGG) (R14) by NMR; the RNA 14-mer forms an intra-strand parallel quadruplex with a G-tetrad and a hexad, in which a G-tetrad core is augmented by association of two A residues. The quadruplex further forms a dimer through stacking interaction between the hexads. In order to obtain insight into the difference between RNA and DNA quadruplexes, we synthesized the corresponding DNA 14-mer, d(GGAGGTTTTGGAGG) (D14), and examined its properties and structure by CD, gel electrophoresis, and NMR. K+ ions increased the thermal stability of both R14 and D14 structures. The binding affinity of K+ ions to R14 was much higher than that to D14. The CD and gel electrophoretic studies suggest that D14 forms a quadruplex entirely different from that of R14 in the presence of K+ ions; two molecules of D14 form a quadruplex with both antiparallel and parallel strand alignments and with diagonal loops at both ends of the stacked G-tetrads. The NMR study also gave results that are consistent with such structure: alternate glycosidic conformation, 5'G(syn)-G(anti)3', and characteristic chemical shift data observed for many quadruplexes containing diagonal TTTT loops.     

Impaired bone fracture healing in matrix metalloproteinase-13 deficient mice

Vascular and cellular invasion into the cartilage is a critical step in the fracture healing. Matrix metalloproteinase-13 (MMP-13) is a member of the zinc-dependent endopeptidase family and plays an important role in remodeling of extracellular matrix. Therefore we investigated the possible involvement of MMP-13 in a murine model of stabilized bone fracture healing. Repair of the fracture in MMP-13 deficient (MMP-13(-/-)) mice was significantly delayed and characterized by a retarded cartilage resorption in the fracture callus. Immunohistochemistry indicated severe defects in vascular penetration and chondroclast recruitment to the fracture callus in MMP-13(-/-) mice. Consistent with the observations, the chondrocyte pellets cultured from the MMP13(-/-) mice exhibited diminished angiogenic activities when the pellets were co-cultured with endothelial cells. These results suggest that MMP-13 is crucial to the process of angiogenesis during healing of fracture, especially in the cartilage resorption process.     

Glucose Deprivation Induces G2/M Transition-Arrest and Cell Death in N-GlcNAc2-Modified Protein-Producing Renal Carcinoma Cells

Some cancer cells can survive under glucose deprivation within the microenvironment of a tumor. Recently, we reported that N-linked (β-N-acetylglucosamine)₂ [N-GlcNAc₂]-modified proteins induce G2/M arrest and cell death under glucose deprivation. Here, we investigated whether such a response to glucose deprivation contributes to the survival of renal cell carcinomas, which are sensitive to nutritional stress. Specifically, we analyzed seven renal carcinoma cell lines. Four of these cell lines produced N-GlcNAc₂-modified proteins and led G2/M-phase arrest under glucose deprivation, leading to cell death. The remaining three cell lines did not produce N-GlcNAc₂-modified proteins and undergo G1/S-phase arrest under glucose deprivation, leading to survival. The four dead cell lines displayed significant up-regulation in the UDP-GlcNAc biosynthesis pathway as well as increased phosphorylation of p53, which was not observed in the surviving three cell lines. In addition, the four dead cell lines showed prolonged up-regulated expression of ATF3, which is related to unfolded protein response (UPR), while the surviving three cell lines showed only transient up-regulation of ATF3. In this study, we demonstrated that the renal carcinoma cells which accumulate N-GlcNAc₂-modified proteins under glucose deprivation do not survive with abnormaly prolonged UPR pathway. By contrast, renal carcinoma cells that do not accumulate N-GlcNAc₂-modified proteins under these conditions survive. Morover, we demonstrated that buformin, a UPR inhibitor, efficiently reduced cell survival under conditions of glucose deprivation for both sensitive and resistant phenotypes. Further studies to clarify these findings will lead to the development of novel chemotherapeutic treatments for renal cancer.     

Mice lacking Ran binding protein 1 are viable and show male infertility

The small GTPase Ran plays important roles in multiple aspects of cellular function. Maximal RanGAP activity is achieved with the aid of RanBP1 and/or presumably of RanBP2. Here, we show that RanBP1-knockout mice are unexpectedly viable, and exhibit male infertility due to a spermatogenesis arrest, presumably caused by down-regulation of RanBP2 during spermatogenesis. Indeed, siRNA-mediated depletion of RanBP2 caused severe cell death only in RanBP1-deficient MEFs, indicating that simultaneous depletion of RanBP1 and RanBP2 severely affects normal cell viability. Collectively, we conclude that the dramatic decrease in "RanBP" activity impairs germ cell viability and affects spermatogenesis decisively in RanBP1-knockout mice.     

Histological Characterization of the Tumorigenic “Peri-Necrotic Niche” Harboring Quiescent Stem-Like Tumor Cells in Glioblastoma

Background

Characterization of the niches for stem-like tumor cells is important to understand and control the behavior of glioblastomas. Cell-cycle quiescence might be a common mechanism underlying the long-term maintenance of stem-cell function in normal and neoplastic stem cells, and our previous study demonstrated that quiescence induced by hypoxia-inducible factor (HIF)-1α is associated with a high long-term repopulation capacity of hematopoietic stem cells. Based on this, we examined human astrocytoma tissues for HIF-1α-regulated quiescent stem-like tumor cells as a candidate for long-term tumorigenic cells and characterized their niche histologically.

Methods

Multi-color immunohistochemistry was used to visualize HIF-1α-expressing (HIF-1α⁺) quiescent stem-like tumor cells and their niche in astrocytoma (WHO grade II–IV) tissues. This niche was modeled using spheroids of cultured glioblastoma cells and its contribution to tumorigenicity was evaluated by sphere formation assay.

Results

A small subpopulation of HIF-1α⁺ quiescent stem-like tumor cells was found in glioblastomas but not in lower-grade astrocytomas. These cells were concentrated in the zone between large ischemic necroses and blood vessels and were closer to the necrotic tissues than to the blood vessels, which suggested that a moderately hypoxic microenvironment is their niche. We successfully modeled this niche containing cells of HIF-1α⁺ quiescent stem-like phenotype by incubating glioblastoma cell spheroids under an appropriately hypoxic condition, and the emergence of HIF-1α⁺ quiescent stem-like cells was shown to be associated with an enhanced sphere-forming activity.

Conclusions

These data suggest that the “peri-necrotic niche” harboring HIF-1α⁺ quiescent stem-like cells confers a higher tumorigenic potential on glioblastoma cells and therefore may be a therapeutic target to control the behavior of glioblastomas.     

Meichroacidin containing the membrane occupation and recognition nexus motif is essential for spermatozoa morphogenesis

Meichroacidin (MCA) is a highly hydrophilic protein that contains the membrane occupation and recognition nexus motif. MCA is expressed during the stages of spermatogenesis from pachytene spermatocytes to mature sperm development and is localized in the male meiotic metaphase chromosome and sperm flagellum. MCA sequences are highly conserved in Ciona intestinalis, Cyprinus carpio, and mammals. To investigate the physiological role of MCA, we generated MCA-disrupted mutant mice; homozygous MCA mutant males were infertile, but females were not. Sperm was rarely observed in the caput epididymidis of MCA mutant males. However, little to no difference was seen in testis mass between wild-type and mutant mice. During sperm morphogenesis, elongated spermatids had retarded flagellum formation and might increase phagocytosis by Sertoli cells. Immunohistochemical analysis revealed that MCA interacts with proteins located on the outer dense fibers of the flagellum. The testicular sperm of MCA mutant mice was capable of fertilizing eggs successfully via intracytoplasmic sperm injection and generated healthy progeny. Our results suggest that MCA is essential for sperm flagellum formation and the production of functional sperm.     

Improved Method for Detection of Vibrio parahaemolyticus in Seafood

We have developed a new, effective procedure for detecting Vibrio parahaemolyticus in seafoods using enrichment and plating onto a chromogenic agar medium. Samples were cultured in salt Trypticase soy broth, which is a nonselective medium, and then a portion of the culture was cultured with salt polymyxin broth, which is a selective medium for V. parahaemolyticus. This two-step enrichment was more effective than the one-step enrichment in salt polymyxin broth alone. The enrichment cultures were then plated onto a new chromogenic agar containing substrates for beta-galactosidase. The V. parahaemolyticus colonies developed a purple color on this growth medium that distinguished them from other related bacterial strains. V. parahaemolyticus was isolated more frequently from naturally contaminated seafood samples using the chromogenic agar than thiosulfate citrate bile salts sucrose agar medium, which is currently used for the isolation of V. parahaemolyticus. Our findings suggest that this new enrichment and isolation scheme is more sensitive and accurate for identifying V. parahaemolyticus in seafood samples than previously used methods.     

Construction of a β-glucosidase expression system using the multistress-tolerant yeast Issatchenkia orientalis

We demonstrate the value of the thermotolerant yeast Issatchenkia orientalis as a candidate microorganism for bioethanol production from lignocellulosic biomass with the goal of consolidated bioprocessing. The I. orientalis MF-121 strain is acid tolerant, ethanol tolerant, and thermotolerant, and is thus a multistress-tolerant yeast. To express heterologous proteins in I. orientalis, we constructed a transformation system for the MF-121 strain and then isolated the promoters of TDH1 and PGK1, two genes that were found to be strongly expressed during ethanol fermentation. As a result, expression of β-glucosidase from Aspergillus aculeatus could be achieved with I. orientalis, demonstrating successful heterologous gene expression in I. orientalis for the first time. The transformant could convert cellobiose to ethanol under acidic conditions and at high temperature. Simultaneous saccharification and fermentation (SSF) was performed with the transformant, which produced 29 g l⁻¹ of ethanol in 72 h at 40°C even without addition of β-glucosidase when SSF was carried out in medium containing 100 g l⁻¹ of microcrystalline cellulose and a commercial cellulase preparation. These results suggest that using a genetically engineered thermotolerant yeast such as I. orientalis in SSF could lead to cost reduction because less saccharification enzymes are required.