An integrated database of the ascidian, Ciona intestinalis: towards functional genomics

An integrated genome database is essential for future studies of functional genomics. In this study, we update cDNA and genomic resources of the ascidian, Ciona intestinalis, and provide an integrated database of the genomic and cDNA data by extending a database published previously. The updated resources include over 190,000 ESTs (672,396 in total together with the previous ESTs) and over 1,000 full-insert sequences (6,773 in total). In addition, results of mapping information of the determined scaffolds onto chromosomes, ESTs from a full-length enriched cDNA library for indication of precise 5'-ends of genes, and comparisons of SNPs and indels among different individuals are integrated into this database, all of these results being reported recently. These advances continue to increase the utility of Ciona intestinalis as a model organism whilst the integrated database will be useful for researchers in comparative and evolutionary genomics.     

Distribution of a novel protein AgK114 expression in the normal tissues of adult mice: dual expression of AgK114 and growth hormone in anterior pituitary cells

The novel antigen K114 (AgK114) has been previously identified in normal hamster skin, and its expression has been up-regulated accompanying tissue damages of the skin, although there is no information on its biological functions. To determine the physiological role of AgK114, we prepared anti-mouse AgK114 monoclonal antibody and studied its tissue distribution in healthy adult mice by immunocytochemistry. A widespread and unique expression of AgK114 peptide was found in the selected organs of various systems (hair follicle cells and sebaceous gland of skin, ciliated epithelial cells of trachea and bronchial tube, striated portion of submandibular gland, distal convoluted tubule cells of kidney, ciliated epithelial cells of oviduct, medulla of adrenal gland and anterior lobe of pituitary gland). Interestingly, dual expression of AgK114 peptide and growth hormone in somatotrophs was found in anterior lobe of pituitary gland by double immunocytochemistry. AgK114 peptide was expressed widely in many regionally well-defined cellular systems in various peripheral tissues, suggesting that AgK114 peptide may have some roles of physiological functions in these organs. The data from our current study have provided a rationale for further studies of functional roles of AgK114 peptide in a variety of organs or tissues under physiological conditions.     

Alternative male mating behaviors dependent on relative body size in captive oval squid Sepioteuthis lessoniana (Cephalopoda, Loliginidae)

We observed the reproductive behavior of the oval squid Sepioteuthis lessoniana in captivity. The male used three different mating behaviors: male-parallel (MP), male-upturned (MU) and sneaking. Male competition over females frequently occurred before and during the female egg-laying period, and the outcome of most fights depended on male body size. Larger males guarded their partners from other males and performed MP mating during the egg-laying period of the paired females. In contrast, there was no pairing and mate guarding in MU mating and sneaking, which were adopted by smaller subordinate males as alternative tactics outside female egg-laying period and during the period, respectively. MP matings were 95% successful, but more than half of MU matings were unsuccessful. Higher mating success in MP mating was achieved through pairing, whereas males in MU mating were less successful because mating attempts without pair formation were often foiled by escape of the female. Sneaking was successful in all cases but occurred less frequently. Spermatophores were attached at the opening of the oviduct in MP mating, whereas they were attached around the female buccal membrane in MU mating and sneaking. Considering the route of egg transportation, higher fertilization success can be expected in MP mating because of the advantageous location of the attached spermatophores. Our results suggest that MP mating is used by larger, paired males during the female egg-laying period, and that MU mating and sneaking are alternative tactics adopted by smaller, subordinate males. These alternative mating behaviors would be conditional strategy dependent on relative body size, because some individual males displayed both MP and MU mating behaviors.     

Morphological examination during in vitro cartilage formation by human mesenchymal stem cells

The formation of the skeleton through endochondral ossification is one of the most complex processes in development. One approach to resolving this complexity is to examine simplified systems. In vitro cartilage formation by mesenchymal stem cells (MSCs) is observed when the cells are cultured as a micromass. Several studies have confirmed the molecular events, showing the usefulness of these cells as a differentiation model. We have elucidated the process of cartilage formation in MSCs from the morphological point of view by light and transmission electron microscopy and immunohistochemical examination. The morphology of the MSCs changed from spherical to spindle-shaped, and the cells aggregated and formed junctional complexes during Day 1. At Day 7, three layers were observed. The superficial zone consisted of several layers of elongated cells with junctional complexes. The middle zone was composed of apoptotic bodies, and the deep zone was occupied by chondrocyte-like cells excreting extracellular matrices. At Day 14, the middle zone had disappeared, and the chondrocyte-like cells in the deep zone were detected within cartilage lacuna. They were covered by cartilage matrices containing collagen types I, II, and X and chondroitin sulfate. By Day 21, the outer layer consisting of spindle-shaped cells had disappeared in places. As the pellet grew, the outer layer seemed to be unable to stretch to maintain a constant covering around the pellet. Our findings have thus revealed that MSCs change their morphology depending upon their microenvironment during differentiation. In vitro cartilage formation by MSCs makes it possible to clarify the detailed morphological events that occur during chondrogenesis. S. Ichinose and I. Sekiya contributed equally to this study     

Prediction of circulatory equilibrium in response to changes in stressed blood volume

Accurate prediction of cardiac output (CO), left atrial pressure (PLA), and right atrial pressure (PRA) is a prerequisite for management of patients with compromised hemodynamics. In our previous study (Uemura et al. Am J Physiol Heart Circ Physiol 286: H2376-H2385, 2004), we demonstrated a circulatory equilibrium framework, which permits the prediction of CO, PLA, and PRA once the venous return surface and integrated CO curve are known. Inasmuch as we also showed that the surface can be estimated from single-point CO, PLA, and PRA measurements, we hypothesized that a similar single-point estimation of the CO curve would enable us to predict hemodynamics. In seven dogs, we measured the PLA-CO and PRA-CO relations and derived a standardized CO curve using the logarithmic function CO = SL[ln(PLA - 2.03) + 0.80] for the left heart and CO = SR[ln(PRA - 2.13) + 1.90] for the right heart, where SL and SR represent the preload sensitivity of CO, i.e., pumping ability, of the left and right heart, respectively. To estimate the integrated CO curve in each animal, we calculated SL and SR from single-point CO, PLA, and PRA measurements. Estimated and measured CO agreed reasonably well. In another eight dogs, we altered stressed blood volume (-8 to +8 ml/kg of reference volume) under normal and heart failure conditions and predicted the hemodynamics by intersecting the surface and the CO curve thus estimated. We could predict CO [y = 0.93x + 6.5, r2 = 0.96, standard error of estimate (SEE) = 7.5 ml.min(-1).kg(-1)], PLA (y = 0.90x + 0.5, r2= 0.93, SEE = 1.4 mmHg), and PRA (y = 0.87x + 0.4, r2= 0.91, SEE = 0.4 mmHg) reasonably well. In conclusion, single-point estimation of the integrated CO curve enables accurate prediction of hemodynamics in response to extensive changes in stressed blood volume.     

Gene therapy for chronic myocardial ischemia using platelet-derived endothelial cell growth factor in dogs

Platelet-derived endothelial cell growth factor (PD-ECGF), also known as thymidine phosphorylase (TP), has been reported to possess angiogenic activity and to inhibit apoptosis. This study was performed to determine whether PD-ECGF/TP can be used to ameliorate chronic myocardial ischemia. Myocardial ischemia was created in 40 mongrel dogs by placement of an ameroid constrictor on the proximal left anterior descending coronary artery (LAD). Plasmid vector encoding human PD-ECGF/TP cDNA (pCIhTP group; n = 12), empty vector pCI (pCI group; n = 12), or saline (Saline group; n = 12) was directly injected into the LAD territory 3 wk after ameroid constrictor implantation. Myocardial blood flow was detected using PET at baseline, 3 wk after ameroid constrictor implantation, and 2 wk after therapeutic treatment. At the end of the experiment, the hearts were isolated for biological and histological analysis. In the pCIhTP group, the transfected heart strongly expressed PD-ECGF/TP. The size of the infarct was smaller in the pCIhTP group than in the pCI or Saline group. The number of apoptotic myocardial cells was decreased in the pCIhTP group compared with the control groups based on triple immunohistochemical staining for von Willebrand factor, alpha-actin smooth muscle cells, and single-strand DNA. The level of proapoptotic protein Bax markedly decreased in the pCIhTP group compared with the other groups. Double immunohistochemical staining for von Willebrand factor and alpha-actin smooth muscle cells demonstrated that angiogenesis and arteriogenesis occurred, and paralleled the changes in myocardial blood flow and myocardial function in the pCIhTP group. We conclude that genetic approaches using PD-ECGF/TP to target the myocardium are effective for alleviating chronic myocardial ischemia.     

Transgenic prolactin-/- mice: effect of trauma-hemorrhage on splenocyte functions

Prolactin (PRL) is involved in the regulation of immune functions under normal and pathological conditions. Trauma-hemorrhage (T-H) produces profound immunosuppression in male mice but not in proestrus female mice. Administration of PRL in males after T-H, however, restores immune functions. In this study, PRL^+/+ and transgenic (PRL^–/–) male and female mice were used to assess immune suppression after T-H and to determine the reasons for the hormone's beneficial effect. In vitro lymphoproliferation assay with Nb2 cells showed complete absence of PRL in the circulation of the transgenic PRL^–/– mice of both sexes, whereas very high levels of the hormone were detected in the wild-type PRL^+/+ mice of both sexes. Moreover, T-H resulted in the appearance of significant levels of the hormone in circulation, but only in PRL^+/+ mice. Splenocyte proliferation in male PRL^–/– mice was significantly lower than in PRL^+/+ mice after T-H. Marginal differences between PRL^+/+ and PRL^–/– mice were observed in the release of IL-2 and IFN- by splenocytes, while the release of IL-10 was significantly higher in PRL^–/– than in PRL^+/+ mice. A significant observation of our study is the release of a 25-kDa protein in the concanavalin A-stimulated splenocytes of male PRL^+/+ and PRL^–/– mice that was active in the in vitro lymphoproliferation assay with Nb2 cells. It is unlikely that this protein is PRL because it is also present in the splenocyte extracts of PRL^–/– transgenic mice. Nonetheless, because control of lymphoid cell proliferation is considered one of the characteristics of the immune system, the local release of this protein may be significant in the differences observed in splenocyte cytokine release after T-H in wild-type as well as transgenic mice. Nb2 cells; cytokines; immune functions     

Enhanced expression of a novel dioxygenase during the early developmental stage of tomato fruit

Most previous efforts to isolate genes that are expressed during fruit development have focused on fruit ripening. As a result, information is lacking on fruit genes that are specifically expressed at early developmental stages. Using a cDNA subtraction technique, we isolated fruit-specific genes that are expressed during the cell expansion phase of tomato (Lycopersicon esculentum Mill) fruit development. One of the isolated cDNAs, LeODD, is transiently expressed 15 days after flowering in a nearly fruit-specific manner during the initial period of cell expansion. Southern blot analysis indicated that LeODD is encoded by a single gene. LeODD is homologous to 2-oxoglutarate-dependent dioxygenase genes, and the key amino acid residues in the binding sites for ferrous iron and 2-oxoglutarate are completely conserved. The amino acid sequence identity between LeODD and other 2-oxoglutarate-dependent dioxygenases is relatively low, suggesting that LeODD is a novel enzyme of this family. Another of the isolated cDNAs, LeGLO2, is also highly expressed at 15 days after flowering. LeGLO2 is thought to be a novel glycolate oxidase isoform that functions in fruit. 2-Oxoglutarate, the cosubstrate of LeODD, could be supplied by a LeGLO2-mediated glycolate pathway in immature fruit. The coordinate expression of LeODD and LeGLO2 may play a role in the biosynthesis of a metabolite, such as a plant hormone or secondary metabolite, that is required during the initial period of the cell expansion phase of fruit development.