Tomoo Sawada

Effects of cytoskeletal inhibitors on ooplasmic segregation and microtubule organization during fertilization and early development in the ascidian Molgula occidentalis

The effects of microtubule and microfilament inhibitors on ooplasmic segregation and microtubule organization were examined during fertilization, parthenogenetic activation, and early development in the ascidian Molgula occidentalis. At fertilization the egg cortex contracts as the first phase movement and shortly after mitochondria migrate as the myoplasmic crescent develops in the second phase. The microtubule inhibitors colcemid and nocodazole inhibit the second phase, but not the first phase, of ooplasmic segregation. The microfilament inhibitor cytochalasin E has the reciprocal effect of inhibiting the first, but not the second, phase. It appears that sperm may initially bind at any site on the egg surface and that the contractile activities at the first phase and during polar body formation occur independent of the microtubule system. Since the second phase migration occurs as the sperm astral microtubules assemble and since microtubule, but not microfilament, inhibitors arrest this aspect of ooplasmic segregation, microtubules appear necessary for mitochondrial migration. These results demonstrate that the two phases of ascidian ooplasmic segregation are mediated by different systems, the first by microfilaments and the second by microtubules. The microtubule and microfilament systems appear to operate independent of one another and their combined actions result in the completion of ooplasmic segregation. A model is proposed in which the cortical contraction following fertilization is important not only as the motive force for the first phase movement but also as a method to unite the myoplasm with the entering sperm which can initially bind anywhere on the egg surface. The association between myoplasmic components and the growing sperm aster would ensure that the migration and the spatial distribution of myoplasm in the second phase results in the formation of the myoplasmic crescent.

FABP7 expression in normal and stab-injured brain cortex and its role in astrocyte proliferation

Reactive gliosis, in which astrocytes as well as other types of glial cells undergo massive proliferation, is a common hallmark of all brain pathologies. Brain-type fatty acid-binding protein (FABP7) is abundantly expressed in neural stem cells and astrocytes of developing brain, suggesting its role in differentiation and/or proliferation of glial cells through regulation of lipid metabolism and/or signaling. However, the role of FABP7 in proliferation of glial cells during reactive gliosis is unknown. In this study, we examined the expression of FABP7 in mouse cortical stab injury model and also the phenotype of FABP7-KO mice in glial cell proliferation. Western blotting showed that FABP7 expression was increased significantly in the injured cortex compared with the contralateral side. By immunohistochemistry, FABP7 was localized to GFAP+ astrocytes (21% of FABP7+ cells) and NG2+ oligodendrocyte progenitor cells (62%) in the normal cortex. In the injured cortex there was no change in the population of FABP7+/NG2+ cells, while there was a significant increase in FABP7+/GFAP+ cells. In the stab-injured cortex of FABP7-KO mice there was decrease in the total number of reactive astrocytes and in the number of BrdU+ astrocytes compared with wild-type mice. Primary cultured astrocytes from FABP7-KO mice also showed a significant decrease in proliferation and omega-3 fatty acid incorporation compared with wild-type astrocytes. Overall, these data suggest that FABP7 is involved in the proliferation of astrocytes by controlling cellular fatty acid homeostasis.

Inflammatory and Anti-inflammatory Cytokines Regulate the Recovery from Sublethal X Irradiation in Rat Thymus

Abstract Mizutani, N., Fujikura, Y., Wang, Y-H., Tamechika, M., Tokuda, N., Sawada, T. and Fukumoto, T. Inflammatory and Anti-inflammatory Cytokines Regulate the Recovery from Sublethal X Irradiation in Rat Thymus. Radiat. Res. 157, 281 – 289 (2002). We investigated the regeneration of rat thymus after sublethal X irradiation (6 Gy). The number of thymocytes was much lower on day 3 after irradiation, and many apoptotic cells were observed. However, by day 5, there had been a rapid proliferation of thymocytes. Since cytokines are considered to be important regulatory factors in postirradiation recovery, we performed in vivo cytokine assays using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and found serial changes in the cytokine message. The messenger RNA (mRNA) expression of the pro-inflammatory cytokines interleukin 1 beta (Il1b), Il6 and tumor necrosis factor alpha (Tnf) was higher than normal on day 3, lower on day 5, and higher again on day 7. In particular, Tnf was completely absent on day 5 and was expressed again on day 7. Of the anti-inflammatory cytokines Il4, transforming growth factor beta (Tgfb) and Il10, only the Il10 message changed substantially. Il10 expression was very high on day 5 but was completely absent on day 7. Thus the Tnf and Il10 messages were expressed alternately. The changes in the distribution of macrophages detected by the immunohistochemical analysis may be related to the changes in the cytokines. Analysis of cytokine messages in the regenerating thymus in vivo may provide new insights into potential therapies for radiation-induced damage.

Classification and Characterization of Hemocytes in Styela clava

Viable hemocytes of the common tunicate Styela clava are classified into four groups designated as eosinophilic granulocytes, basophilic granulocytes, hyaline cells and lymphocyte-like cells. Eosinophilic granulocytes, actively amoeboid, have large refractive granules that stain with neutral red. Basophilic granulocytes do not stain with neutral red and formed couplets or triplets. Hyaline cells, which often contain phagosomes, have electron-dense small vesicles recognizable only by electron microscopy. Hemoblasts have a characteristic large nucleolus which is visible by light microscopy. Eosinophilic granulocytes and hyaline cells actively ingest yeast particles in vitro. This classification simplifies former ones by correlating electron microscopy, with light microscopy, and viable with fixed hemocytes. Clearly viable tunicate hemocytes can be identified by simple methods. We have provided clear and more accurate descriptions which will lessen the controversy often associated with assigning hemocyte functions in immunodefense responses both in vivo and in vitro.

Dynamics of mitotic apparatus formation and tubulin content during oocyte maturation in starfish

To follow the topo‐temporal behavior of structures containing tubulin and the change in tubulin content during oocyte maturation, starfish oocytes were extracted with a medium containing detergent so that morphological observation and biochemical analysis could be conducted on the same residual oocyte preparation simultaneously. Before 1‐methyladenine (1‐MeAde) stimulation, “pre‐meiotic asters” were observed on the germinal vesicle at the animal pole. 1‐MeAde caused the appearance of distinct asters at the position of the aster precursor. When germinal vesicle breakdown (GVBD) took place, chromosomes were condensed. Chromosome gathering was concurrent with a reduction in the size of nuclear matrix. The mitotic apparatus was first constructed parallel to the cortex and then changed its axis perpendicularly. Fluorescence of tubulin due to indirect immunofluorescence in the cytoplasm other than the mitotic apparatus decreased rapidly along the course of maturation at least up to the first metaphase. Despite these dynamic morphological change, the tubulin content in the whole oocyte and the residual structures, measured by SDS‐PAGE and immunostaining, did not show remarkable (statistically significant) changes through the course of maturation, although the content tended to decrease a little before the second polar body formation and to increase thereafter in the latter.

Fatty acid-binding protein 7 regulates function of caveolae in astrocytes through expression of caveolin-1.

Fatty acid‐binding proteins (FABPs) bind and solubilize long‐chain fatty acids, controlling intracellular lipid dynamics. FABP7 is expressed by astrocytes in the developing brain, and suggested to be involved in the control of astrocyte lipid homeostasis. In this study, we sought to examine the role of FABP7 in astrocytes, focusing on plasma membrane lipid raft function, which is important for receptor‐mediated signal transduction in response to extracellular stimuli. In FABP7‐knockout (KO) astrocytes, the ligand‐dependent accumulation of Toll‐like receptor 4 (TLR4) and glial cell‐line‐derived neurotrophic factor receptor alpha 1 into lipid raft was decreased, and the activation of mitogen‐activated protein kinases and nuclear factor‐κB was impaired after lipopolysaccharide (LPS) stimulation when compared with wild‐type astrocytes. In addition, the expression of caveolin‐1, not cavin‐1, 2, 3, caveolin‐2, and flotillin‐1, was found to be decreased at the protein and transcriptional levels. FABP7 re‐expression in FABP7‐KO astrocytes rescued the decreased level of caveolin‐1. Furthermore, caveolin‐1‐transfection into FABP7‐KO astrocytes significantly increased TLR4 recruitment into lipid raft and tumor necrosis factor‐α production after LPS stimulation. Taken together, these data suggest that FABP7 controls lipid raft function through the regulation of caveolin‐1 expression and is involved in the response of astrocytes to the external stimuli. GLIA 2015;63:780–794

Fatty Acid Binding Protein 3 Is Involved in n–3 and n–6 PUFA Transport in Mouse Trophoblasts

BACKGROUND Low placental fatty acid (FA) transport during the embryonic period has been suggested to result in fetal developmental disorders and various adult metabolic diseases, but the molecular mechanism by which FAs are transported through the placental unit remains largely unknown. OBJECTIVE The aim of this study was to examine the distribution and functional relevance of FA binding protein (FABP), a cellular chaperone of FAs, in the mouse placenta. METHODS We clarified the localization of FABPs and sought to examine their function in placental FA transport through the phenotypic analysis of Fabp3-knockout mice. RESULTS Four FABPs (FABP3, FABP4, FABP5, and FABP7) were expressed with spatial heterogeneity in the placenta, and FABP3 was dominantly localized to the trophoblast cells. In placentas from the Fabp3-knockout mice (both sexes), the transport coefficients for linoleic acid (LA) were significantly reduced compared with those from wild-type mice by 25% and 44% at embryonic day (E) 15.5 and E18.5, respectively, whereas those for α-linolenic acid (ALA) were reduced by 19% and 17%, respectively. The accumulation of LA (18% and 27% at E15.5 and E18.5) and ALA (16% at E15.5) was also significantly less in the Fabp3-knockout fetuses than in wild-type fetuses. In contrast, transport and accumulation of palmitic acid (PA) were unaffected and glucose uptake significantly increased by 23% in the gene-ablated mice compared with wild-type mice at E18.5. Incorporation of LA (51% and 52% at 1 and 60 min, respectively) and ALA (23% at 60 min), but not PA, was significantly less in FABP3-knockdown BeWo cells than in controls, whereas glucose uptake was significantly upregulated by 51%, 50%, 31%, and 33% at 1, 20, 40, and 60 min, respectively. CONCLUSIONS Collectively FABP3 regulates n-3 (ω-3) and n-6 (ω-6) polyunsaturated FA transport in trophoblasts and plays a pivotal role in fetal development.

Identification of FABP7 in fibroblastic reticular cells of mouse lymph nodes

Fatty acids and their metabolites regulate immune cell function. The present study was undertaken to examine the detailed distribution of fatty acid binding proteins (FABPs), the cytosolic chaperones of fatty acids, in mouse peripheral immune organs. Using immunohistochemistry, FABP7 was localized to the alpha-smooth muscle actin (SMA)+ fibroblastic reticular cells, which construct the stromal reticula in the T cell areas of the peripheral lymph nodes and spleen. Immunoelectron microscopy showed that FABP7+ cells enclosed the collagen fibers, forming a conduit system, which transport lymph and associated low-molecular-mass proteins. In contrast, FABP5+ cells were distributed throughout the lymph node and contained well-developed lysosome and phagocytic materials within the cytoplasm. The mesenteric lymph nodes of FABP7 knockout mice showed normal histological features, but the percentage of CD4+ cells was significantly increased compared with that in wild-type mice. These data indicate that FABP7 may be involved in T cell homeostasis, possibly by modulating lipid metabolism in fibroblastic reticular cells within the peripheral lymph nodes.

Fatty acid-binding protein regulates LPS-induced TNF-α production in mast cells

There has been increasing evidence for the involvement of fatty acid-binding proteins (FABPs) in the cytokine production of macrophages and dendritic cells probably through the control of cellular lipid metabolism and signal transduction. Since mast cells (MCs) are recently shown to be involved in immune response through modification of cytokine production, it is possible that some FABPs could also be involved in the immune response of MCs. In this study, we found that epidermal-type FABP (E-FABP) was expressed in murine bone marrow-derived MCs (BMMCs). Using BMMCs from genetically E-FABP-null mutated mice, we demonstrated that E-FABP in BMMCs plays a key role in the production of TNF-alpha following lipopolysaccharide (LPS) stimulation. In the in vivo septic peritonitis model (cecal ligation and puncture model), E-FABP-null mice showed a significantly increased mortality compared to wild-type mice. However, no significant difference in antigen-induced cytokine production was observed between wild-type and E-FABP-null BMMCs, and systemic anaphylaxis was equally induced in vivo in both wild-type and E-FABP-null mice. These results suggest that E-FABP is specifically involved in the LPS-induced cytokine production of MCs, and could play a role in the host-defense against bacterial infection, possibly through regulation of TNF-alpha production.

Semiquantitative Detection of Cytokine Messages in X-Irradiated and Regenerating Rat Thymus

Abstract Adachi, Y., Tokuda, N., Sawada, T. and Fukumoto, T. Semiquantitative Detection of Cytokine Messages in X-Irradiated and Regenerating Rat Thymus. Radiat. Res. 163, 400– 407 (2005). We investigated the expression of cytokine mRNA derived from thymocytes or thymic epithelial cells in X-irradiated (8 Gy) and recovering rat thymuses, according to our previous observation (Mizutani et al., Radiat. Res. 157, 281–289, 2002). The changes in mRNA expression level of interleukin 2 (Il2), Il4, tumor necrosis factor α (Tnf), interferon γ (Ifng), and transforming growth factor β (Tgfb) were examined. The mRNA expression of Il2 and Il4 decreased from day 5 to day 14 after irradiation. Thereafter, the expression level of Il2 mRNA recovered to normal control levels; however, the expression of Il4 mRNA tended toward significantly low levels. Tnf mRNA expression decreased on day 5 after irradiation and then showed a gradual increase back to normal control levels. Tgfb mRNA expression did not change significantly. Ifng mRNA expression was transiently enhanced from day 11 to day 14. The mRNA expression levels of Il10 increased significantly from day 3 to day 7 after irradiation. In addition, the mRNA expression of thymic epithelial cell-derived Il7 showed a transient decrease on day 3; however, then it showed a continuous increase from day 5 to day 21, finally reaching twice the normal control levels after X irradiation. These observations suggest that the expression of cytokine messages in the irradiated thymus changed significantly and did not return to normal for a long time after 8 Gy irradiation.