Cryopreservation of spermatozoa of a transgenic mouse]

Spermatozoa of a homozygous transgenic mouse, in which the firefly luciferase gene was expressed under the control of beta-actin promoter, were frozen at -196 degrees C. One fourth of the frozen sperm was later thawed and used for in vitro fertilization. Thirty-six of 65 oocytes (55.4%) developed to the 2-cell stage. All the 2-cell embryos were transferred to the oviducts of pseudopregnant recipients and 23 young (63.9%, 23/36) were born. All of young analyzed carried the transgene and showed the luciferase gene expression.     

Effects of Hydrostatic Pressure on Carcinogenic Properties of Epithelia

The relationship between chronic inflammation and cancer is well known. The inflammation increases the permeability of blood vessels and consequently elevates pressure in the interstitial tissues. However, there have been only a few reports on the effects of hydrostatic pressure on cultured cells, and the relationship between elevated hydrostatic pressure and cell properties related to malignant tumors is less well understood. Therefore, we investigated the effects of hydrostatic pressure on the cultured epithelial cells seeded on permeable filters. Surprisingly, hydrostatic pressure from basal to apical side induced epithelial stratification in Madin-Darby canine kidney (MDCK) I and Caco-2 cells, and cavities with microvilli and tight junctions around their surfaces were formed within the multi-layered epithelia. The hydrostatic pressure gradient also promoted cell proliferation, suppressed cell apoptosis, and increased transepithelial ion permeability. The inhibition of protein kinase A (PKA) promoted epithelial stratification by the hydrostatic pressure whereas the activation of PKA led to suppressed epithelial stratification. These results indicate the role of the hydrostatic pressure gradient in the regulation of various epithelial cell functions. The findings in this study may provide clues for the development of a novel strategy for the treatment of the carcinoma.     

Retinol induces platelet aggregation via activation of phospholipase A2

All-trans-retinol induced aggregation of rabbit platelets, and this effect could be inhibited by a cyclooxygenase inhibitor and a thromboxane A2 (TXA2) receptor antagonist, indicating an essential role for endogenously produced TXA2. We found a two-phase arachidonic acid release in retinol-stimulated platelets. The first phase was induced by the action of retinol alone and not inhibited by TXA2 receptor antagonist. The second phase was induced via synergistic action of retinol and initially generated small amount of TXA2, and was inhibited by the antagonist. Moreover, we discussed that the arachidonic acid release may be mediated by the action of phospholipase A2.     

Polyclonal Antibody Production in Murine Spleen Cells Induced by Staphylococcus

Polyclonal plaque-forming cell (PFC) responses in murine spleen cells induced by Staphylococcus aureus and S. epidermidis were studied. Injection of Balb/c mice with S. aureus strain 248βH resulted in the generation of anti-trinitrophenyl (TNP) and anti-sheep red blood cell PFC in their spleens. Cultures of Balb/c spleen cells in the presence of S. aureus 248βH, Cowan I, or a protein A-deficient mutant yielded many anti-TNP PFC. The larger the number of organisms that were added to the cultures, the better was the PFC response. Both living and killed organisms, were capable of inducing the response, but an excess of living 248βH organisms in the cultures abrogated the response. All of the organisms (12 strains of S. aureus and 11 strains of S. epidermidis) freshly isolated from patients had the ability to induce the polyclonal PFC response in cell cultures. These organisms stimulated cultured C3H/HeJ mouse spleen cells, which were unresponsive to bacterial lipopolysaccharide (LPS). Cultured cells from the spleens of athymic nu/nu mice also responded to these organisms, and the number of PFC in nu/nu cell cultures was always greater than that in nu/+ cells prepared from a haired litter mate. Moreover, the responses of nu/nu spleen cell cultures to which nylon wool column-filtered splenic nu/+ T cells were added were lower than expected. These findings suggest that the polyclonal PFC response to staphylococci is thymus independent, but that the magnitude of the response is regulated by mature T cells. Cultures of macrophage-depleted spleen cells responded to the organisms to an extent similar to that of the control. The 248βH organisms were less capable of stimulating spleen cells of 2-week-old mice (i.e., early maturing B cells) than LPS. However, spleen cells from adult (7-week-old) and aged (9-month-old) mice responded well to both the organisms and LPS. Previous sensitization with the organisms in vivo did not affect any polyclonal responses of spleen cells in vitro to either the organisms or LPS. The role of staphylococcal protein A in the polyclonal PFC response to staphylococci is discussed.     

Electron microscope studies on the vegetative cellular life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture I. Some characteristics of changes in subcellular structures during the cell cycle, especially in formation of giant mitochondria

Changes in subcellular structures during the entire vegetativecell cycle of Chlamydomonas reinhardi Dangeard in synchronousculture were followed with an electron microscope. Giant mitochondriaof various shapes were temporarily formed, probably by fusionof smaller mitochondria, in the algal cells at an intermediatestage of the growth phase of the cell cycle. Formation of giantmitochondria was accompanied by a marked decrease in the oxygen-uptakeactivity of cells. Giant mitochondria divided into smaller formsconcurrently with a re-increase in the oxygen-uptake activityof cells. Some characteristics of changes in the structuresof chloroplast, the nucleus, the endoplasmic reticula, flagellaand dictyosomes are described. ¹ This work was reported in part at the 35th Annual Meetingof the Botanical Society of Japan, October 1970. (Received October 13, 1971; )     

Fractionation of chromosomal proteins of pea seedlings using procedures which avoid denaturation

Chromatin was prepared from the buds and cotyledons of Alaskapea seedlings. The dissociated chromosomal components in thepresence of 2 M NaCl and 5 M urea were completely fractionatedinto DNA and proteins with a Bio-Gel A50 column. The proteinswere recovered by (NH₄)₂SO₄ and further fractionated into histonesand non-histone proteins using a Bio-Rex 70(Na⁺) column. Thedifference in the ratios of histones to non-histone proteinsbefore and after chromatography with the Bio-Rex 70 was lessthan 10%. The histones and non-histone proteins thus preparedshowed typical protein absorption spectra. Polyacrylamide gelelectrophoresis of histones showed that the histone compositionsin buds and cotyledon were similar, but the amount of HI histoneswas a little less in cotyledons than in buds. Unlike histones,non-histone proteins fractionated by SDS-polyacrylamide gelelectrophoresis indicated distinct differences between the twotissues. Buds had more heterogeneous non-histone proteins, atleast 13 polypeptides, than cotyledons did. On the other hand,non-histone proteins of cotyledons showed less heterogeneityand lacked proteins of high molecular weight which were foundin buds. (Received May 6, 1976; )     

MiR-376c Down-Regulation Accelerates EGF-Dependent Migration by Targeting GRB2 in the HuCCT1 Human Intrahepatic Cholangiocarcinoma Cell Line

MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.     

Organic Acid Metabolism in Aluminum-Phosphate Utilizing Cells of Carrot (Daucus carota L.)

Carbohydrate metabolism in Al-phosphate utilizing cells of carrot[designated as IPG, Koyama et al. (1992) Plant Cell Physiol.33: 171], which grow normally in Al-phosphate medium accompaniedby citrate excretion, was investigated. The excretion of citratewas strongly related to the availability of sucrose in medium,indicating that citrate excretion was severely limited by sucrosein medium. The ratio of the amount of carbon in the excretedcitrate to the consumed sucrose, was significantly higher inIPG cells than in wild-type cells. When 50% of the sucrose inthe medium was consumed, the ratio was 0.6% for the IPG cellsand 0.2% the wild-type cells. Under these conditions, IPG cellsshowed altered citrate synthesis metabolism, which resultedin increased citrate production. Specific activity of mitochondrialcitrate synthase was higher in IPG cells than in wild-type cells,whereas the activity of cytosolic NADP-specific isocitrate dehydrogenasewas lower in IPG cells than in wild-type cells. (Received August 27, 1998; Accepted February 21, 1999)     

Cross-sectional Area-corrected Compression Modulus of Food Gel

The linearity of the stress-strain relationship for food gel is limited to a very narrow range of the strain (usually less than 0.1 as a Cauchy measure). The reason is thought due to the change in cross-sectional area of the gel upon deformation. In this report, the cross-sectional area was approximately corrected of the compressed gel on the assumption that the gel expanded uniformly without changing its volume upon compression. In cases when the initial Young’s modulus was calculated from the thus-corrected area for some food gels, the linearity was increased for a wider range of strain.