Novel Repetitive Structures, Deviant Protein-Encoding Sequences andUnidentified ORFs in the Mitochondrial Genome of the BrachiopodLingula anatina

Complete sequence determination of the brachiopod Lingula anatina mtDNA (28,818 bp) revealed an organization that is remarkably atypical for an animal mt-genome. In addition to the usual set of 37 animal mitochondrial genes, which make up only 57% (16,555 bp) of the entire sequence, the genome contains lengthy unassigned sequences. All the genes are encoded in the same DNA strand, generally in a compact way, whereas the overall gene order is highly divergent in comparison with known animal mtDNA. Individual genes are generally longer and deviate considerably in sequence from their homologues in other animals. The genome contains two major repeat regions, in which 11 units of unassigned sequences and six genes (atp8, trnM, trnQ, trnV, and part of cox2 and nad2) are found in repetition, in the form of nested direct repeats of unparalleled complexity. One of the repeat regions contains unassigned repeat units dispersed among several unique sequences, novel repetitive structure for animal mtDNAs. Each of those unique sequences contains an open reading frame for a polypeptide between 80 and 357 amino acids long, potentially encoding a functional molecule, but none of them has been identified with known proteins. In both repeat regions, tRNA genes or tRNA gene-like sequences flank major repeated units, supporting the view that those structures play a role in the mitochondrial gene rearrangements. Although the intricate repeated organization of this genome can be explained by recurrent tandem duplications and subsequent deletions mediated by replication errors, other mechanisms, such as nonhomologous recombinations, appear to explain certain structures more easily.     

LIP19, a basic region leucine zipper protein, is a Fos-like molecular switch in the cold signaling of rice plants

The rice low-temperature-induced lip19 gene encodes a 148-amino-acid basic region/leucine zipper (bZIP) protein, termed LIP19. In this study we characterized LIP19 and showed that it lacks the usual ability of bZIP proteins to homodimerize and to bind DNA, as does the Fos protein in mammals. Using a yeast two-hybrid system, the cDNA clones whose products interact with LIP19 were screened. This search revealed a clone termed OsOBF1 (Oryza sativa OBF1) that encodes a new bZIP protein (OsOBF1). This protein forms a homodimer and binds to the hexamer motif sequence (5'-ACGTCA-3'). The protein-protein interaction in homo- and hetero-combinations between LIP19 and OsOBF1 was confirmed in vitro and in planta. LIP19 and OsOBF1 most likely interact with each other more strongly than OsOBF1 interacts with itself, and the resulting heterodimer binds to the C/G hybrid sequence but not to the hexamer sequence. Whereas the expression patterns of lip19 and OsOBF1 in response to low temperatures were totally opposite, the locations of their expression were almost identical. Based upon the presented data, we propose a model describing the low-temperature signal switching mediated by LIP19 in rice.     

Promotive effect of macrophage colony-stimulating factor on long-term engraftment of murine hematopoietic stem cells

Large ex vivo expansion of hematopoietic stem cells (HSCs) sufficient for use in clinical applications has not been achieved, although the influence of some cytokines including SCF, IL-11, Flt3-L, and TPO for this purpose has been reported. We present evidence for an indirect effect of macrophage colony-stimulating factor (M-CSF) on expansion of murine HSCs. Fresh Lin(-/low) cells were isolated from Ly5.1 mouse bone marrow and cultured with or without M-CSF in the presence of SCF + IL-11 + Flt3-L or SCF + IL-11 + TPO for 6 days. The expanded cells were harvested and transplanted into lethally irradiated Ly5.2 recipients with competitor cells. Culture of Lin(-/low) cells with M-CSF significantly enhanced long-term engraftment. When the more enriched HSC populations of Lin(-/low) c-Kit(+) Sca-1(+) cells were used as a source of HSCs, such a promotive effect was not observed, in agreement with negative expression of the M-CSF receptor (c-Fms). However, co-culture with Lin(-/low) c-Fms(+) resulted in a significant increase of long-term engraftment. These results suggested that M-CSF is an indirect stimulator for ex vivo expansion of HSCs in the presence of SCF, IL-11, Flt3-L, and TPO. These observations provide new directions for ex vivo expansion and insight into new engraftment regulation through M-CSF signaling.     

Gastric inhibitory polypeptide modulates adiposity and fat oxidation under diminished insulin action

Gut hormone gastric inhibitory polypeptide (GIP) stimulates insulin secretion from pancreatic β-cells upon ingestion of nutrients. Inhibition of GIP signaling prevents the onset of obesity and consequent insulin resistance induced by high-fat diet. In this study, we investigated the role of GIP in accumulation of triglycerides into adipocytes and in fat oxidation peripherally using insulin receptor substrate (IRS)-1-deficient mice and revealed that IRS-1^−/−GIPR^−/− mice exhibited both reduced adiposity and ameliorated insulin resistance. Furthermore, increased gene expression of CD36 and UCP2 in liver, and increased expression and enzyme activity of 3-hydroxyacyl-CoA dehydrogenase in skeletal muscle of IRS-1^−/−GIPR^−/− mice might contribute to the lower respiratory quotient and the higher fat oxidation in light phase. These results suggest that GIP plays a crucial role in switching from fat oxidation to fat accumulation under the diminished insulin action as a potential target for secondary prevention of insulin resistance.     

Pyrazolo pyrimidine-type inhibitors of SRC family tyrosine kinases promote ovarian steroid-induced differentiation of human endometrial stromal cells in vitro

Reversible protein tyrosine phosphorylation, coordinately controlled by protein tyrosine kinases and phosphatases, is a critical element in signal transduction pathways regulating a wide variety of biological processes, including cell growth, differentiation, and tumorigenesis. We have previously reported that c-Src belonging to the Src family tyrosine kinase (SFK) becomes dephosphorylated at tyrosine 530 (Y530) and thereby activated during progestin-induced differentiation of human endometrial stromal cells (i.e., decidualization). In this study, to elucidate the role of decidual c-Src activation, we examined whether 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), both potent and selective SFK inhibitors, affected the ovarian steroid-induced decidualization in vitro. Unexpectedly, PP1 paradoxically increased the kinase activity of decidual c-Src together with dephosphorylation of Y530 in the presence of ovarian steroids. Concomitantly, PP1 enhanced morphological and functional decidualization, as determined by induction of decidualization markers, such as insulin-like growth factor binding protein-1 and prolactin. PP2 also advanced decidualization along with up-regulation of the active form of c-Src whose Y-530 was dephosphorylated. In contrast to PP1 and PP2, herbimycin A, a tyrosine kinase inhibitor with less specificity for SFKs, showed little enhancing effect on the expression of both IGFBP-1 and active c-Src. These results suggest that SFKs, including c-Src, may play a significant role in stromal cell differentiation, providing a clue for a possible therapeutic strategy to modulate endometrial function by targeting signaling pathway(s) involving SFKs.     

Multiple sampling for increasing the diagnostic sensitivity of nipple aspirate fluid for atypical cytology

OBJECTIVE: To determine if repeated collection of nipple aspirate fluid (NAF) can improve the diagnostic sensitivity for cytologic atypia, a marker of increased risk of breast cancer. STUDY DESIGN: Two hundred sixty-seven women without known breast disease volunteered for NAF cytology at 5 6-month intervals over 2 years. NAF samples were prepared on Millipore filters (Millipore Filter Corp., Bedford, Massachusetts, U.S.A.) and stained with a modified Papanicolaou method. Fluid availability and cellular abnormalities were evaluated for each collection attempt. Cellular findings were classified as benign, hyperplasia or atypia. RESULTS: NAF was obtained from 178 women (66.6%) at the first visit and from an additional 15, 10, 2 and 4 women at visits 2, 3, 4 and 5, respectively, for a cumulative total of 78.2% by visit 5. The number of women yielding NAF containing hyperplastic or atypical epithelial cells was determined at each visit. Hyperplastic cells were found in 34 (19.1%) at visit 1 and in an additional 20, 10, 5 and 4 women at visits 2, 3, 4 and 5, respectively. Atypical epithelial cells were present in 12 (6.7%) women at the initial visit and in an additional 11, 7, 5 and 1 women at visits 2, 3, 4 and 5, respectively, for a cumulative percent of 18.2 at visit 5. NAF could not be obtained from 58 women at any visit. CONCLUSION: These findings suggest that an optimum collection method for NAF cytology should consist of at least 3 or 4 separate fluid aspiration attempts. Reviewing repeated multiple samples instead of 1 increases the number of women who can be evaluated and the likelihood of detecting cytologic atypia.     

Chondrogenic differentiation of human mesenchymal stem cells cultured in a cobweb-like biodegradable scaffold

Human mesenchymal stem cells (MSCs) were cultured in vitro in a cobweb-like biodegradable polymer scaffold: a poly(dl-lactic-co-glycolic acid)-collagen hybrid mesh in serum-free DMEM containing TGF-beta3 for 1-10 weeks. The cells adhered to the hybrid mesh, distributed evenly, and proliferated to fill the spaces in the scaffold. The ability of the cells to express gene encoding type I collagen decreased, whereas its ability to express type II collagen and aggrecan increased. Histological examination by HE staining indicated that the cells showed fibroblast morphology at the early stage and became round after culture for 4 weeks. The cartilaginous matrices were positively stained by safranin O and toluidine blue. Immunostaining with anti-type II collagen and anti-cartilage proteoglycan showed that type II collagen and cartilage proteoglycan were detected around the cells. In addition, a homogeneous distribution of cartilaginous extracellular matrices was detected around the cells. These results suggest the chondrogenic differentiation of the mesenchymal stem cells in the hybrid mesh. The PLGA-collagen hybrid mesh enabled the aggregation of mesenchymal stem cells and provided a promotive microenvironment for the chondrogenic differentiation of the MSCs.     

The importance of adipose-derived stem cells and vascularized tissue regeneration in the field of tissue transplantation

The importance of stem cells in regenerating or repairing damaged or diseased tissues is well established, but three factors have to be considered in employing stem cells clinically. The first is how to harvest, handle, and multiply them non-invasively, easily, and effectively. From this standpoint, adipose-derived stem cells are considered to be the best to work with among mesenchymal stem cells; since they were first reported in 2001, their pluripotency, proliferative efficiency, and low donor morbidity have been amply confirmed. The second factor is how to differentiate stem cells into the required cells and use them effectively to construct three-dimensional tissues; here, tissue-specific scaffolds and signaling systems are essential. The third factor is how to ensure survival of the differentiated cells and regenerated tissues. Regenerated tissues need to contain vascular systems to allow both the tissues themselves and the differentiated cells to survive. Thus, we believe that the vascularization of regenerated tissues will be an important field of research in the near future. In this paper, we focus on adipose-derived stem cells and vascularized tissue regeneration within the context of tissue transplantation.     

Elucidation of the speciation history of three sister species of crown-of-thorns starfish (Acanthaster spp.) based on genomic analysis

The crown-of-thorns starfish (COTS) is a coral predator that is widely distributed in Indo-Pacific Oceans. A previous phylogenetic study using partial mitochondrial sequences suggested that COTS had diverged into four distinct species, but a nuclear genome-based analysis to confirm this was not conducted. To address this, COTS species nuclear genome sequences were analysed here, sequencing Northern Indian Ocean (NIO) and Red Sea (RS) species genomes for the first time, followed by a comparative analysis with the Pacific Ocean (PO) species. Phylogenetic analysis and ADMIXTURE analysis revealed clear divergences between the three COTS species. Furthermore, within the PO species, the phylogenetic position of the Hawaiian sample was further away from the other Pacific-derived samples than expected based on the mitochondrial data, suggesting that it may be a PO subspecies. The pairwise sequentially Markovian coalescent model showed that the trajectories of the population size diverged by region during the Mid-Pleistocene transition when the sea-level was dramatically decreased, strongly suggesting that the three COTS species experienced allopatric speciation. Analysis of the orthologues indicated that there were remarkable genes with species-specific positive selection in the genomes of the PO and RS species, which suggested that there may be local adaptations in the COTS species.