A conserved non-reproductive GnRH system in chordates

Gonadotropin-releasing hormone (GnRH) is a neuroendocrine peptide that plays a central role in the vertebrate hypothalamo-pituitary axis. The roles of GnRH in the control of vertebrate reproductive functions have been established, while its non-reproductive function has been suggested but less well understood. Here we show that the tunicate Ciona intestinalis has in its non-reproductive larval stage a prominent GnRH system spanning the entire length of the nervous system. Tunicate GnRH receptors are phylogenetically closest to vertebrate GnRH receptors, yet functional analysis of the receptors revealed that these simple chordates have evolved a unique GnRH system with multiple ligands and receptor heterodimerization enabling complex regulation. One of the gnrh genes is conspicuously expressed in the motor ganglion and nerve cord, which are homologous structures to the hindbrain and spinal cord of vertebrates. Correspondingly, GnRH receptor genes were found to be expressed in the tail muscle and notochord of embryos, both of which are phylotypic axial structures along the nerve cord. Our findings suggest a novel non-reproductive role of GnRH in tunicates. Furthermore, we present evidence that GnRH-producing cells are present in the hindbrain and spinal cord of the medaka, Oryzias latipes, thereby suggesting the deep evolutionary origin of a non-reproductive GnRH system in chordates.     

The direct interaction of phospholipase C-gamma 1 with phospholipase D2 is important for epidermal growth factor signaling

The epidermal growth factor (EGF) receptor has an important role in cellular proliferation, and the enzymatic activity of phospholipase C (PLC)-gamma1 is regarded to be critical for EGF-induced mitogenesis. In this study, we report for the first time a phospholipase complex composed of PLC-gamma1 and phospholipase D2 (PLD2). PLC-gamma1 is co-immunoprecipitated with PLD2 in COS-7 cells. The results of in vitro binding analysis and co-immunoprecipitation analysis in COS-7 cells show that the Src homology (SH) 3 domain of PLC-gamma1 binds to the proline-rich motif within the Phox homology (PX) domain of PLD2. The interaction between PLC-gamma1 and PLD2 is EGF stimulation-dependent and potentiates EGF-induced inositol 1,4,5-trisphosphate (IP(3)) formation and Ca(2+) increase. Mutating Pro-145 and Pro-148 within the PX domain of PLD2 to leucines disrupts the interaction between PLC-gamma1 and PLD2 and fails to potentiate EGF-induced IP(3) formation and Ca(2+) increase. However, neither PLD2 wild type nor PLD2 mutant affects the EGF-induced tyrosine phosphorylation of PLC-gamma1. These findings suggest that, upon EGF stimulation, PLC-gamma1 directly interacts with PLD2 and this interaction is important for PLC-gamma1 activity.     

15-deoxy-delta 12,14-prostaglandin J2 and laminar fluid shear stress stabilize c-IAP1 in vascular endothelial cells

Laminar shear stress strongly inhibits vascular endothelial cell apoptosis by unknown mechanisms. We reported that shear stress stimulates endothelial cells to produce 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) by elevating the expression level of lipocalin-type prostaglandin D synthase. To investigate the role of 15d-PGJ2 produced in the vascular wall, we examined the effect of 15d-PGJ2 on endothelial cell apoptosis. We induced apoptosis in human umbilical vein endothelial cells (HUVECs) by growth factor deprivation. 15d-PGJ2 strongly inhibited DNA ladder formation, nuclear fragmentation, and caspase-3-like activity in HUVECs. To elucidate the mechanism by which 15d-PGJ2 inhibits endothelial cell apoptosis, we examined expression of the inhibitor of apoptosis proteins (IAP) cellular-IAP1 (c-IAP1), c-IAP2, x-linked IAP, and survivin in HUVECs. In parallel with the inhibition of apoptosis, 15d-PGJ2 elevated the expression level of c-IAP1 protein in a dose- and time-dependent manner without changing the mRNA level. Laminar shear stress also induced c-IAP1 expression. Chase experiments with the use of cycloheximide revealed that 15d-PGJ2 and shear stress both inhibited the proteolytic degradation of c-IAP1 protein. These results suggested that 15d-PGJ2 inhibits endothelial cell apoptosis through, at least in part, c-IAP1 protein stabilization. This mechanism might be involved in the antiapoptotic effect of laminar shear stress.     

Regulation of Brassica rapa chloroplast proliferation in vivo and in cultured leaf disks

Summary. To understand the regulatory mechanisms of chloroplast proliferation, chloroplast replication was studied in cultured leaf disks cut from plants of 25 species. In leaf disks from Brassica rapa var. perviridis, the number of chloroplasts per cell increased remarkably in culture. We examined chloroplast replication in this plant in vivo and in culture media with and without benzyladenine, a cytokinin. In whole plants, leaf cells undergo two phases from leaf emergence to full expansion: an early proliferative stage, in which mitosis occurs, and a differentiational stage after mitosis has diminished. During the proliferative stage, chloroplast replication keeps pace with cell division. In the differentiational phase, cell division ceases but chloroplast replication continues for two or three more cycles, with the number of chloroplasts per cell reaching about 60. In the leaf disks, the number of chloroplasts per cell increased from about 18 to 300 without benzyladenine, and to over 600 with benzyladenine, indicating that this cytokinin enhances chloroplast replication in cultured tissue. We also studied changes in ploidy and cell volume between in vivo cells and cells grown in culture with and without benzyladenine. Ploidy and cell volume increased in a manner very similar to that of the number of chloroplasts, suggesting a relationship between these phenomena.Correspondence and reprints: Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan.     

Wnt2b controls retinal cell differentiation at the ciliary marginal zone

The ciliary marginal zone of the vertebrate retina contains undifferentiated progenitor cells that continue to proliferate and add new neurons and glia peripherally during the embryonic stages - even after the formation of a functional retina. To understand the molecular mechanism that controls the prolonged progenitor cell proliferation in the ciliary marginal zone, we employed a candidate molecule approach, focusing on Wnt2b (formerly know as Wnt13), which is expressed in the marginal most tip of the retina. Frizzled 4 and 5, seven-pass transmembrane Wnt receptors, were expressed in the peripheral and central part of the retina, respectively. LEF1, a downstream Wnt signaling component, was expressed at high levels in the ciliary marginal zone with expression gradually decreasing towards the central retina. The LEF1-expressing region, which is where Wnt signaling is supposedly activated, expressed a set of molecular markers that are characteristic of the progenitor cells in the ciliary marginal zone. Overexpression of Wnt2b by use of in ovo electroporation in the central retina inhibited neuronal differentiation and induced the progenitor cell markers. Blocking of the Wnt downstream signaling pathway by a dominant-negative LEF1 inhibited proliferation of the cells in the marginal area, which resulted in their premature neuronal differentiation. The progenitor cells in the ciliary marginal zone differentiated into all the neuronal and glial cell types when cultured in vitro, and they proliferated for a longer period than did centrally located progenitor cells that underwent a limited number of cell divisions. In addition, the proliferation of these progenitor cells was promoted in the presence of Wnt2b. These results suggest that Wnt2b functions to maintain undifferentiated progenitor cells in the ciliary marginal zone, and thus serves as a putative stem cell factor in the retina.     

Identification and characterization of the SSB1 locus involved in symptom development by Spring beauty latent virus infection in Arabidopsis thaliana

The natural variation of Arabidopsis thaliana in response to a bromovirus, Spring beauty latent virus (SBLV), was examined. Of 63 Arabidopsis accessions tested, all were susceptible when inoculated with SBLV, although there was a large degree of variation in symptom development. Most accessions, including Columbia (Col-0), were symptomless or developed only mild symptoms, but four accessions, including S96, showed severe symptoms of SBLV infection. Genetic analysis suggested that the difference in the responses of Col-0 and S96 to SBLV was controlled by a single semidominant locus. We have designated this locus SSB1 (symptom development by SBLV infection). By using genetic markers, SSB1 was mapped to chromosome IV. The patterns of distribution and accumulation of SBLV in sensitive accessions were similar to those in the insensitive accessions. In addition, symptom development in S96 by SBLV infection was critically interrupted by the presence of the NahG gene, which encodes salicylic acid (SA) hydroxylase. These data suggest that symptom development in A. thaliana controlled by SSB1 is independent of the efficiency of SBLV multiplication and is dependent on SA signaling.     

Systemic administration of IL-18 promotes diabetes development in young nonobese diabetic mice

IL-18 is now identified as a pleiotropic cytokine that acts as a cofactor for both Th1 and Th2 cell development. Type 1 diabetes is considered a Th1-type autoimmune disease, and to date, the suppressive effect of exogenous IL-18 on the development of diabetes has been reported in 10-wk-old nonobese diabetic (NOD) mice. In the present study we administered exogenous IL-18 systemically in 4-wk-old NOD mice using i.m. injection of the IL-18 expression plasmid DNA (pCAGGS-IL-18) with electroporation. Contrary to previous reports, the incidence of diabetes development was significantly increased in NOD mice injected with pCAGGS-IL-18 compared with that in control mice. Systemic and pancreatic cytokine profiles deviated to a Th1-dominant state, and the the frequency of glutamic acid decarboxylase-reactive IFN-gamma-producing CD4(+) cells was also high in the IL-18 group. Moreover, it was suggested that the promoting effect of IL-18 might be associated with increased peripheral IL-12, CD86, and pancreatic IFN-inducible protein-10 mRNA expression levels. In conclusion, we demonstrate here that IL-18 plays a promoting role as an enhancer of Th1-type immune responses in diabetes development early in the spontaneous disease process, which may contribute to elucidating the pathogenesis of type 1 diabetes.     

The origin and organization of cortical microtubules during the transition between M and G1 phases of the cell cycle as observed in highly synchronized cells of tobacco BY-2

The organization of microtubules (MTs) during the transition from the M phase to the G₁ phase of the cell cycle was followed in highly synchronized suspension-cultured cells ofNicotiana tabacum L. (tobacco BY-2) by sequential treatment of cells with aphidicolin and propyzamide. Short MTs were first formed in the perinuclear regions at the expense of phragmoplasts, but when these short MTs elongated to reach the cell cortex, they grew parallel to the long axis and towards the distal end of the cells. As soon as, or shortly before the tips of elongated MTs reached the distal end, transverse cortical MTs were formed in the region proximal to the division plane. Thereafter, almost all cells retained cortical MTs which were transversely orientated to the long axis of cells and could be observed in the G₁ phase. Thus, in the organization of cortical MTs, there are two steps that have been overlooked thus far. This novel observation provides a new scheme for the organization of cortical MTs, which could unify two contrasting hypotheses, i.e. organization in the perinuclear regions versus that in the cell cortex. These observations are discussed in relation to the microtubule-organizing center of plant cells.     

Studies on the fungal flora in the lung of human necropsy cases. A critical survey in connection with the pathogenesis of opportunistic fungus infections

An ecological study was carried out on the fungal flora in the human lung of 159 autopsy cases. Fungi were isolated from 129 cases (81.1%). Filamentous fungi consisting of 918 strains were isolated from 113 cases, and the dominant genera were Aspergillus and Penicillium. Yeasts were isolated from 58 cases, and the dominant genus appeared to be Candida.With morbid anatomical study, the incidence of the fungus isolation was examined from various points of view. It was concluded that even a healthy respiratory parenchyma of the human lung cannot be assumed as aseptic. However, only a few numbers of a few genera of air-born fungi were isolated.Special stress was laid on the exposure of the respiratory parenchyma of the human lung to air-born fungi in connection with the pathogenesis of opportunistic fungus infections.