Dmp1-deficient mice display severe defects in cartilage formation responsible for a chondrodysplasia-like phenotype

Understanding the molecular mechanisms by which cartilage formation is regulated is essential toward understanding the physiology of both embryonic bone development and postnatal bone growth. Although much is known about growth factor signaling in cartilage formation, the regulatory role of noncollagenous matrix proteins in this process are still largely unknown. In the present studies, we present evidence for a critical role of DMP1 (dentin matrix protein 1) in postnatal chondrogenesis. The Dmp1 gene was originally identified from a rat incisor cDNA library and has been shown to play an important role in late stage dentinogenesis. Whereas no apparent abnormalities were observed in prenatal bone development, Dmp1-deficient (Dmp1(-/-)) mice unexpectedly develop a severe defect in cartilage formation during postnatal chondrogenesis. Vertebrae and long bones in Dmp1-deficient (Dmp1(-/-)) mice are shorter and wider with delayed and malformed secondary ossification centers and an irregular and highly expanded growth plate, results of both a highly expanded proliferation and a highly expanded hypertrophic zone creating a phenotype resembling dwarfism with chondrodysplasia. This phenotype appears to be due to increased cell proliferation in the proliferating zone and reduced apoptosis in the hypertrophic zone. In addition, blood vessel invasion is impaired in the epiphyses of Dmp1(-/-) mice. These findings show that DMP1 is essential for normal postnatal chondrogenesis and subsequent osteogenesis.     

Semiquantitative detection of cytokine messages in X-irradiated and regenerating rat thymus

We investigated the expression of cytokine mRNA derived from thymocytes or thymic epithelial cells in X-irradiated (8 Gy) and recovering rat thymuses, according to our previous observation (Mizutani et al., Radiat. Res. 157, 281-289, 2002). The changes in mRNA expression level of interleukin 2 (Il2), Il4, tumor necrosis factor alpha (Tnf), interferon gamma (Ifng), and transforming growth factor beta (Tgfb) were examined. The mRNA expression of Il2 and Il4 decreased from day 5 to day 14 after irradiation. Thereafter, the expression level of Il2 mRNA recovered to normal control levels; however, the expression of Il4 mRNA tended toward significantly low levels. Tnf mRNA expression decreased on day 5 after irradiation and then showed a gradual increase back to normal control levels. Tgfb mRNA expression did not change significantly. Ifng mRNA expression was transiently enhanced from day 11 to day 14. The mRNA expression levels of Il10 increased significantly from day 3 to day 7 after irradiation. In addition, the mRNA expression of thymic epithelial cell-derived Il7 showed a transient decrease on day 3; however, then it showed a continuous increase from day 5 to day 21, finally reaching twice the normal control levels after X irradiation. These observations suggest that the expression of cytokine messages in the irradiated thymus changed significantly and did not return to normal for a long time after 8 Gy irradiation.     

Structure of model peptides based on Nephila clavipes dragline silk spidroin (MaSp1) studied by 13C cross polarization/magic angle spinning NMR

To obtain detailed structural information for spider dragline spidroin (MaSp1), we prepared three versions of the consensus peptide GGLGGQGAGAAAAAAGGAGQGGYGGLGSQGAGR labeled with 13C at six different sites. The 13C CP/MAS NMR spectra were observed after treating the peptides with different reagents known to alter silk protein conformations. The conformation-dependent 13C NMR chemical shifts and peak deconvolution were used to determine the local structure and the fractional compositions of the conformations, respectively. After trifluoroacetic acid (solvent)/diethyl ether (coagulant) treatment, the N-terminal region of poly-Ala (PLA) sequence, Ala8 and Ala10, adopted predominantly the alpha-helix with a substantial amount of beta-sheet. The central region, Ala15, Ala18, and Leu26, and C-terminal region, Ala31, of the peptide were dominated by either 3(1)-helix or alpha-helix. There was no indication of beta-sheet, although peak broadening indicates that the torsion angle distribution is relatively large. After 9 M LiBr/dialysis treatment, three kinds of conformation, beta-sheet, random coil, and 3(1)-helix, appeared, in almost equal amounts of beta-sheet and random coil conformations for Ala8 and Ala10 residues and distorted 3(1)-helix at the central region of the peptide. In contrast, after formic acid/methanol and 8 M urea/acetonitrile treatments, all of the local structure tends to beta-sheet, although small amounts of random coil are also observed. The peak pattern of the Ala Cbeta carbon after 8 M urea/acetonitrile treatment is similar to the corresponding patterns of silk fiber from Bombyx mori and Samia cynthia ricini. We also synthesized a longer 13C-labeled peptide containing two PLA blocks and three Gly-rich blocks. After 8 M urea/acetonitrile treatment, the conformation pattern was closely similar to that of the shorter peptide.     

Molecular cloning and characterization of Trypanosoma vivax alternative oxidase (AOX) gene, a target of the trypanocide ascofuranone

Trypanosoma vivax causes nagana disease in cattle. Since T. vivax is transmitted not only by tsetse flies but also by other biting flies (non-cyclic transmission), the parasite has been distributed to and has had a significant economic impact on wide geographical areas, including Africa and South America. Our previous study on Trypanosoma brucei brucei showed that the trypanosome alternative oxidase (TAO, TbAOX) is a promising target of chemotherapy. For this reason, we also have cloned the T vivax AOX (TvAOX) gene and characterized the recombinant enzyme. The deduced amino acid sequence (328 a.a.) of TvAOX shares 76% identity with TbAOX and contains the diiron-coordination motifs (-E-, -EXXH-) that are conserved among AOXs. The Km of recombinant TvAOX (rTvAOX) expressed in Escherichia coli for ubiquinol (87.0 +/- 0.54 microM) was significantly lower than the value for recombinant TbAOX (rTbAOX) (714 +/- 4.5 microM). Ascofuranone, the most potent inhibitor of TbAOX, was a competitive inhibitor of rTvAOX with a Ki value (0.40 +/- 0.00 nM) significantly lower than that for rTbAOX (1.29 +/- 0.00 nM). The non-cyclic transmission ability of T. vivax and the in vivo chemotherapeutic efficacy of ascofuranone against T. vivax and T. b. brucei infection are discussed in terms of these Km and Ki values.     

Genotypic variation among lineages of Trypanosoma cruzi and its geographic aspects

Isozyme analysis with 18 enzyme loci was conducted on 146 isolates of Trypanosoma cruzi from Mexico, Guatemala, Colombia, Ecuador, Peru, Brazil, Bolivia, Paraguay and Chile. Forty-four different MLGs (groups of isolates with identical multilocus genotypes) were identified and a phylogeny was constructed. The phylogenetic tree consisted of two main groups (T. cruzi I, T. cruzi II), and the latter was further divided into two subgroups (T. cruzi IIa, T. cruzi IIb–e). Evidence of hybridization between different MLGs of T. cruzi II was found, which means that genetic exchanges seem to have occurred in South American T. cruzi. On the other hand, the persistence of characteristic T. cruzi I and T. cruzi II isozyme patterns in single small villages in Bolivia and Guatemala suggested that genetic exchange is very rare between major lineages. A significant difference in genetic diversity was shown between T. cruzi I and T. cruzi II from several indices of population genetics. Two possibilities could explain this genetic variation in the population: differences in evolutionary history and/or different tendencies to exchange genetic material. Broad-scale geographic distributions of T. cruzi I and T. cruzi IIb–e were different; T. cruzi I occurred in Central America and south to Bolivia and Brazil, while T. cruzi IIb–e occurred in the central and southern areas of South America, overlapping with T. cruzi I in Brazil and Bolivia.     

Cilostazol enhances IL-1beta-induced NO production and apoptosis in rat vascular smooth muscle via PKA-dependent pathway

Interleukin-1beta (IL-1beta) stimulates nitric oxide (NO) production and induces apoptosis in several tissues. Cilostazol is a Type 3 phosphodiesterase inhibitor. We investigated whether cilostazol affects IL-1beta-induced NO production and apoptosis in rat vascular smooth muscle cells. Cilostazol (100 nM-10 microM) potentiated NO production triggered by IL-1beta. The mRNA and protein expression of inducible NO synthase was also upregulated by cilostazol. KT5720, an inhibitor of protein kinase A, and N(G)-monomethyl-L-arginine, an inhibitor of NO synthase, abrogated cilostazol-enhanced IL-1beta-stimulated NO production and apoptosis. These results shows that cilostazol potentiates IL-1beta-induced NO production via PKA-pathway and thereafter augments apoptosis via NO-dependent pathway.     

Cloning and characterization of cDNAs for the jasmonic acid-responsive Genes RRJ1 and RRJ2 in suspension-cultured rice cells

Two cDNA clones for jasmonic acid (JA)-responsive genes, RRJ1 and RRJ2, were isolated by differential screening from suspension-cultured rice cells treated with JA for 2 h. The putative RRJ1 protein is completely identical to that of a putative rice cystathionine gamma-lyase, while the putative RRJ2 protein is highly similar in sequence to a rice pyruvate decarboxylase, PDC1.     

Molecular cloning of mouse collectin liver 1

Collectins are members of the superfamily of vertebrate C-type lectins that contain a collagen-like region, and are involved in first-line host defense. We earlier cloned and characterized a new kind of collectin, collectin liver 1 (CL-L1). In this study, we isolated the mouse homologue of CL-L1 encoding 277 amino acid residues; its deduced protein sequence was 88% identical with human CL-L1. Mouse CL-L1 mRNA was expressed mainly in the liver and stomach, but was found also in muscles, testes, intestines, and embryos. In mouse embryos, the level of CL-L1 mRNA gradually increased with embryonic age. In 16-day-old mouse embryos, CL-L1 mRNA was expressed in the liver, amnion, and visceral yolk sac. The mouse CL-L1 gene, Cll1 was found on chromosome 15 in a region syntenic with human chromosome 8q. CL-L1 was a highly conserved protein in mammals, birds, and fish.     

Intercellular trafficking of herpes simplex virus type 2 UL14 deletion mutant proteins

The UL14 gene product of herpes simplex virus is a 32kDa protein expressed late in infection and is a minor component of the virion tegument. We recently showed that the wild-type UL14 protein has heat shock protein (HSP)-like and/or molecular chaperone-like functions. In this study, the intracellular localization of UL14 wild-type and deletion mutant proteins was examined in transfected cells by immunofluorescence. We found that N-terminus deleted but not wild-type/C-terminus deleted mutant proteins showed a significant number of cytoplasmic, multi-cellular stains in transfected Vero cells. The effect was greatly intensified by subjecting cells to heat shock at 43 degrees C, whereas it was obstructed by treatment with the microfilament-disrupting drug cytochalasin D. The staining patterns of UL14 antigen-positive cells after heat shock suggested a cell-to-cell spread of the protein. Although the mechanism is unclear, the phenomenon seems to be an unprecedented type of intercellular trafficking.