Chondrogenesis enhanced by overexpression of sox9 gene in mouse bone marrow-derived mesenchymal stem cells

We investigated chondrogenesis of cell-mediated sox9 gene therapy as a new treatment regimen for cartilage regeneration. pIRES2-EGFP vector containing a full-length mouse sox9 cDNA was transfected into bone marrow-derived mesenchymal stem cells (MSCs) by lipofection and chondrogenic differentiation of these cells was evaluated. In vitro high density micromass culture of these sox9 transfected MSCs demonstrated that a matrix-rich micromass aggregate with EGFP expressing MSCs was positively stained by Alcian blue and type II collagen. Next, sox9 transfected MSCs were loaded into the diffusion chamber and transplanted into athymic mice to analyze in vivo chondrogenesis. A massive tissue formation in about 2mm diameter was visible in the chamber after 4 weeks transplantation. Histological examinations demonstrated that both Alcian blue and type II collagen were positively stained in the extracellular matrix of the mass while type X collagen was not stained. These results indicated that cell-mediated sox9 gene therapy could be a novel strategy for hyaline cartilage damage.     

New linker proteins in phycobilisomes isolated from the cyanobacterium Gloeobacter violaceus PCC 7421

Two new linker proteins were identified by peptide mass fingerprinting in phycobilisomes isolated from the cyanobacterium Gloeobacter violaceus PCC 7421. The proteins were products of glr1262 and glr2806. Three tandem phycocyanin linker motifs similar to CpcC were present in each. The glr1262 product most probably functions as a rod linker connecting phycoerythrin and phycocyanin, while the glr2806 product may function as a rod-core linker. We have designated these two proteins CpeG and CpcJ, respectively. The morphology of phycobilisomes in G. violaceus has been reported to be a bundle-like shape with six rods, consistent with the proposed functions of these linkers.     

A minimal base‐pairing region of a bacterial small RNA SgrS required for translational repression of ptsG mRNA

Escherichia coli SgrS is an Hfq‐binding small RNA that is induced under glucose‐phosphate stress to cause translational repression and RNase E‐dependent rapid degradation of ptsG mRNA encoding the major glucose transporter. A 31‐nt‐long stretch in the 3′ region of SgrS is partially complementary to the translation initiation region of ptsG mRNA. We showed previously that SgrS alone causes translational repression when pre‐annealed with ptsG mRNA by a high‐temperature treatment in vitro. Here, we studied translational repression of ptsG mRNA in vitro by synthetic RNA oligonucleotides (oligos) to define the SgrS region required for translational repression. We first demonstrate that a 31 nt RNA oligo corresponding to the base‐pairing region is sufficient for translational inhibition of ptsG mRNA. Then, we show that RNA oligo can be shortened to 14 nt without losing its effect. Evidence shows that the 14 nt base‐pairing region is sufficient to inhibit ptsG translation in the context of full‐length SgrS in vivo. We conclude that SgrS 168–181 is a minimal base‐pairing region for translational inhibition of ptsG mRNA. Interestingly, the 14 nt oligo efficiently inhibited ptsG translation without the high‐temperature pre‐treatment, suggesting that remodelling of structured SgrS is an important mechanism by which Hfq promotes the base pairing.     

Genome-wide association study identifies HLA-DP as a susceptibility gene for pediatric asthma in Asian populations

Asthma is a complex phenotype influenced by genetic and environmental factors. We conducted a genome-wide association study (GWAS) with 938 Japanese pediatric asthma patients and 2,376 controls. Single-nucleotide polymorphisms (SNPs) showing strong associations (P<1×10⁻⁸) in GWAS were further genotyped in an independent Japanese samples (818 cases and 1,032 controls) and in Korean samples (835 cases and 421 controls). SNP rs987870, located between HLA-DPA1 and HLA-DPB1, was consistently associated with pediatric asthma in 3 independent populations (P _combined = 2.3×10⁻¹⁰, odds ratio [OR] = 1.40). HLA-DP allele analysis showed that DPA1*0201 and DPB1*0901, which were in strong linkage disequilibrium, were strongly associated with pediatric asthma (DPA1*0201: P = 5.5×10⁻¹⁰, OR = 1.52, and DPB1*0901: P = 2.0×10⁻⁷, OR = 1.49). Our findings show that genetic variants in the HLA-DP locus are associated with the risk of pediatric asthma in Asian populations.     

Structure of the rat alpha 2-macroglobulin-coding gene

Rat alpha 2-macroglobulin (alpha 2M) is an acute-phase protein, i.e., produced upon tissue inflammation. Genomic DNA clones covering the entire sequence of the alpha 2M gene were isolated and characterized by restriction mapping. Southern blotting and (partial) DNA sequencing. The rat alpha 2M gene is approx. 50 kb in length and consists of 36 exons ranging in size from 21 to 229 bp. Two functional domains, a bait region and a thiol ester site, are encoded by the exon 18 and 24, respectively. Several possible regulatory signals such as a TPA-inducible enhancer core, an identifier sequence, purine-pyrimidine alternative stretches and viral enhancer core sequences were identified. Several genomic DNA clones which cross-hybridized with the alpha 2M cDNA probe were also identified. Sequence analysis showed that they possessed sequences identical to a part of the rat alpha 1-inhibitor III cDNA and that they had a strikingly similar exon organization to the alpha 2M gene.     

Preliminary genetic characterization of two lineages of black rats (Rattus rattus sensu lato) in Japan, with evidence for introgression at several localities

We conducted a pilot survey of genetic diversity among 37 karyotyped individuals of the black rat Rattus rattus (sensu lato) from six localities on the Japanese Islands, using complete gene sequences of mitochondrial cytochrome b (cyt b) and nuclear interphotoreceptor retinoid binding protein (IRBP). Our sampling included two previously documented karyotypic groups: 'Oceanian' with 2n = 38 and 'Asian' with 2n = 42. Cyt b sequences for most individuals clustered according to their karyotypic groups, with an average between-group divergence of 3.8%. One exception was that individuals from Kagoshima (Kyushu Island) showed 'Asian' karyotypes combined with a cyt b haplotype that differed by a single nucleotide substitution from the haplotype of the 'Oceanian' karyotypic group. Six IRBP haplotypes were identified. They belonged to three distinct IRBP lineages (I-III), with an average inter-lineage divergence of 1%. Among homozygous individuals, these lineages showed good association with the karyotypic groups: IRBP lineage I occurred only with 'Oceanian' karyotypes, while IRBP lineages II and III both occurred with 'Asian' karyotypes. Individuals from Kagoshima all possessed IRBP of 'Asian' lineages, despite the presence of an 'Oceanian' mitochondrial type. The Chichijima population (Ogasawara Islands) featured exclusively 'Asian' karyotypes and cyt b sequences, but various combinations of all three IRBP lineages. The Kagoshima and Chichijima populations thus provide strong evidence of viable hybridization and genetic introgression between the two karyotypic groups, but with variable genetic outcomes. Our results demonstrate the potential of combined analysis of karyotypes and mitochondrial and nuclear gene sequences to elucidate the complex dispersal and population history of the black rat.     

Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles

We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS(97)xS(99)). Surprisingly, serine 97 is not appreciably phosphorylated, whereas serine 99 is only a specific substrate for Akt2 but not Akt1 or Akt3. Although wild-type Synip (WT-Synip) undergoes an insulin-stimulated dissociation from Syntaxin4, the Synip serine 99 to phenylalanine mutant (S99F-Synip) is resistant to Akt2 phosphorylation and fails to display insulin-stimulated Syntaxin4 dissociation. Furthermore, overexpression of WT-Synip in 3T3L1 adipocytes had no effect on insulin-stimulated recruitment of glucose transporter 4 (GLUT4) to the plasma membrane, whereas overexpression of S99F-Synip functioned in a dominant-interfering manner by preventing insulin-stimulated GLUT4 recruitment and plasma membrane fusion. These data demonstrate that insulin activation of Akt2 specifically regulates the docking/fusion step of GLUT4-containing vesicles at the plasma membrane through the regulation of Synip phosphorylation and Synip-Syntaxin4 interaction.