TGF-β mediated FGF10 signaling in cranial neural crest cells controls development of myogenic progenitor cells through tissue-tissue interactions during tongue morphogenesis

Skeletal muscles are formed from two cell lineages, myogenic and fibroblastic. Mesoderm-derived myogenic progenitors form muscle cells whereas fibroblastic cells give rise to the supportive connective tissue of skeletal muscles, such as the tendons and perimysium. It remains unknown how myogenic and fibroblastic cell-cell interactions affect cell fate determination and the organization of skeletal muscle. In the present study, we investigated the functional significance of cell-cell interactions in regulating skeletal muscle development. Our study shows that cranial neural crest (CNC) cells give rise to the fibroblastic cells of the tongue skeletal muscle in mice. Loss of Tgfbr2 in CNC cells (Wnt1-Cre;Tgfbr2^flox/flox) results in microglossia with reduced Scleraxis and Fgf10 expression as well as decreased myogenic cell proliferation, reduced cell number and disorganized tongue muscles. Furthermore, TGF-β2 beads induced the expression of Scleraxis in tongue explant cultures. The addition of FGF10 rescued the muscle cell number in Wnt1-Cre;Tgfbr2^flox/flox mice. Thus, TGF-β induced FGF10 signaling has a critical function in regulating tissue-tissue interaction during tongue skeletal muscle development.     

Identification of Genes Associated with Local Aggressiveness and Metastatic Behavior in Soft Tissue Tumors

Soft tissue tumors represent a group of neoplasia with different histologic and biological presentations varying from benign, locally confined to very aggressive and metastatic tumors. The molecular mechanisms responsible for such differences are still unknown. The understanding of these molecular alterations mechanism will be critical to discriminate patients who need systemic treatment from those that can be treated only locally and could also guide the development of new drugs'' against this tumors. Using 102 tumor samples representing a large spectrum of these tumors, we performed expression profiling and defined differentially expression genes that are likely to be involved in tumors that are locally aggressive and in tumors with metastatic potential. We described a set of 12 genes (SNRPD3, MEGF9, SPTAN-1, AFAP1L2, ENDOD1, SERPIN5, ZWINTAS, TOP2A, UBE2C, ABCF1, MCM2, and ARL6IP5) showing opposite expression when these two conditions were compared. These genes are mainly related to cell-cell and cell-extracellular matrix interactions and cell proliferation and might represent helpful tools for a more precise classification and diagnosis as well as potential drug targets.     

Structural studies of heterochiral DNA/DNA, RNA/RNA, and DNA/RNA duplexes

Using DNA and RNA heptanucleotides containing an unnatural L-nucleotides as well as the complementary strands, effects of the introduction of an L-nucleotide on the structure of DNA/DNA, RNA/RNA, and DNA/RNA duplexes were investigated by circular dichroism experiments and RNase H-mediated RNA strand cleavage reaction. The results suggested that the substitution of the central D-nucleotide with an L-nucleotide in the duplexes causes the significant structural alterations as the duplex structures change to conformations with more B-form similarities.     

Temperature-sensitive mutation in yeast mitochondrial ribosome recycling factor (RRF)

The yeast protein Rrf1p encoded by the FIL1 nuclear gene bears significant sequence similarity to Escherichia coli ribosome recycling factor (RRF). Here, we call FIL1 Ribosome Recycling Factor of yeast, RRF1. Its gene product, Rrf1p, was localized in mitochondria. Deletion of RRF1 leads to a respiratory incompetent phenotype and to instability of the mitochondrial genome (conversion to rho^–/rho⁰ cytoplasmic petites). Yeast with intact mitochondria and with deleted genomic RRF1 that harbors a plasmid carrying RRF1 was prepared from spores of heterozygous diploid yeast. Such yeast with a mutated allele of RRF1, rrf1-L209P, grew on a non-fermentable carbon source at 30 but not at 36°C, where mitochondrial but not total protein synthesis was 90% inhibited. We propose that Rrf1p is essential for mitochondrial protein synthesis and acts as a RRF in mitochondria.     

Construction of a gamete-enriched gene pool and RNAi-mediated functional analysis in Dictyostelium discoideum

Macrocysts in Dictyostelium discoideum possess prototypic features of sexual reproduction and are useful for understanding the basic mechanisms of the reproductive process. Here, we randomly analyzed 1,071 gamete cDNAs, and then constructed a gamete-specific subtraction library, FC-IC. Nucleotide sequences of all 903 FC-IC clones were determined and clustered into 272 independent genes. Expression analysis based on real-time RT-PCR revealed 67 gamete-enriched genes, among which those involved in 'signal transduction' and 'multicellular organization' are prevalent. One of them, FC-IC0003, appeared also to be mating-type specific, and was named gmsA. RNAi-mediated silencing as well as disruption of gmsA reduced the cellular competency for sexual cell fusion, indicating the involvement of this gene in the sexual development of D. discoideum.     

Dermorphin tetrapeptide analogues with 2',6'-dimethylphenylalanine (Dmp) substituted for aromatic amino acids have high mu opioid receptor binding and biological activities

To investigate the value of the 2',6'-dimethylphenylalanine (Dmp) residue as an aromatic amino acid substitution, we prepared analogues of the mu opioid receptor-selective dermorphin tetrapeptide Tyr-D-Arg-Phe-betaAla-NH(2) (YRFB) in which Dmp or its D-isomer replaced Tyr(1) or Phe(3). Replacing Phe(3) with Dmp essentially tripled mu receptor affinity and the receptor's in vitro biological activities as determined with the guinea pig ileum (GPI) assay but did not change delta receptor affinity. Despite an inversion of the D configuration at this position, mu receptor affinity and selectivity remained comparable with those of the L-isomer. Replacing the N-terminal Tyr residue with Dmp produced a slightly improved mu receptor affinity and a potent GPI activity, even though the substituted compound lacks the side chain phenolic hydroxyl group at the N-terminal residue. Dual substitution of Dmp for Tyr(1) and Phe(3) produced significantly improved mu receptor affinity and selectivity compared with the singly substituted analogues. Subcutaneous injection of the two analogues, [Dmp(3)]YRFB and [Dmp(1)]YRFB, in mice produced potent analgesic activities that were greater than morphine in the formalin test. These lines of evidence suggest that the Dmp residue would be an effective aromatic amino acid surrogate for both Tyr and Phe in the design and development of novel opioid mimetics.     

Design and synthesis of 2,6-diprenyl-4-iodophenol TX-1952 with a novel and potent anti-peroxidative activity

The structure-based elucidation of 2,4,6-tri-substituted phenols for their antioxidative and anti-peroxidative effects has been investigated using TX-1952 (2,6-diprenyl-4-iodophenol), TX-1961, TX-1980, BTBP and BHT. In the inhibition of mitochondrial lipid peroxidation, the inhibitory activity of 2,6-di-tert-butyl-4-bromophenol (BTBP) (IC(50)=0.17 microM) was twice as high as that of 2,6-di-tert-butyl-4-methylphenol (BHT) (IC(50)=0.31 microM). This result shows that the 4-halogen group increases inhibitory activity for mitochondrial lipid peroxidation. Besides, TX-1952 (IC(50)=0.60 microM) was the highest inhibitor among 2,6-diprenyl-4-halophenols, followed by TX-1961 (IC(50)=0.93 microM) and TX-1980 (IC(50)=1.2 microM). In 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging experiments, the activity of TX-1952 (IC(0.200)=53.1 microM) was lower than that of BHT (IC(0.200)=33.7 microM) and BTBP (IC(0.200)=16.0 microM), but TX-1952 and BHT showed the same HOMO energy (-8.991 eV). These results suggest that the two prenyl groups at ortho position hinder the phenolic hydrogen abstraction by DPPH radical. These findings demonstrated that TX-1952 was a novel and potent inhibitor for lipid peroxidation.