Water Soluble Pigments Containing Xylose and Glucose in Gametangia of the Green Alga, Bryopsis maxima

Several water-soluble pigments were purified from gametangiaof Bryopsis maxima by liquid chromatography and characterizedby pyridylamination and high-performance anion-exchange chromatography.The structure of the main red pigment is proposed based on thedata of infrared spectrum, Mass spectrum, ¹H and ¹³C NMR spectraand pyridylamino analysis. As a consequence, this pigment containeda tetrapyrrole with phytol and a sugar chain comprised of xyloseand glucose. The sequence of the sugars in the chain was determinedbased on its Mass spectrum. The pigment was similar to chlorophyll-originpigments observed in other plants. No aldehyde group, however,was present at C5 in the open tetrapyrrole chain. (Received August 3, 1994; Accepted November 10, 1994)     

Establishing apoptosis resistant cell lines for improving protein productivity of cell culture

The authors established apoptosis resistant COS–1, myeloma, hybridoma, and Friend leukemia cell lines by genetically engineering cells, aiming at more efficient protein production by cell culture. COS–1 cells, which are most widely used for eukariotic gene expression, were transfected with human bcl–2 gene. Both bcl–2 and mock transfected COS–1 cells were cultured at low (0.2%) serum concentration for 9 days. The final viable cell number of the bcl–2 transfected cells was ninefold of that of the mock transfectants. Both bcl–2 and mock transfectants were further transfected with the vector pcDNA- containing SV40 ori and immunoglobulin gene for transiently expressing protein. The bcl–2 expressing COS–1 cells produced more protein than the mock transfected COS–1 cells after 4 days posttransfection.Mouse myeloma p3-X63-Ag.8.653 cells, which are widely used as the partner for preparing hybridoma, and hybridoma 2E3 cells were transfected with human bcl–2 gene. Both bcl–2 transfected myeloma and hybridoma survived longer than the corresponding original cells in batch culture. The bcl–2 transfected 2E3 cells survived 2 to 4 four days longer in culture, producing 1.5- to 4-fold amount of antibody in comparison with the mock transfectants.Coexpression of bag–1 with bcl–2 improved survival of hybridoma 2E3 cells more than bcl–2 expression alone. The bag–1 and bcl–2 coexpressing cells produced more IgG than the the cells expressing bcl–2 alone.Apoptosis of Friend murine erythroleukemia(F-MEL) cells was suppressed with antisense c-jun expression. The antisense c-jun expressing cells survived 16 days at non-growth state.     

The specific expression of tenascin in the synovial membrane of the temporomandibular joint with internal derangement: An immunohistochemical study

 The expression of tenascin (TN) in the temporomandibular joint (TMJ) disc and synovial membrane was examined in 18 human TMJ samples from patients with internal derangement of the TMJ and ten control specimens by an immunohistological technique using paraffin-embedded tissue and specific anti-human TN monoclonal antibody (RCB-1). The expression of TN was observed in all 28 samples, but it was limited to the walls of blood vessels, the perineurium, and the surface of the TMJ disc. The expression of TN was diffuse in the stroma of mildly hypertrophic synovial membranes and focal in the surface of severely hypertrophic synovial membranes. The clinical symptoms of internal derangement of the TMJ are thought to be related to the degree of synovitis. The present study demonstrates that TN is expressed specifically in the portion of the TMJ synovial membrane affected with internal derangement. Accepted: 17 December 1996     

Induction of T-cell apoptosis by human herpesvirus 6.

The mechanisms of cell death in CD4+ T cells mediated by human herpesvirus 6 (HHV-6) were investigated. The frequency of cell death in the human CD4+ T-cell line JJHAN, which had been inoculated with HHV-6 variant A or B, appeared to be augmented by tumor necrosis factor alpha (TNF-alpha). Agarose gel electrophoresis of DNA from HHV-6-inoculated cells showed DNA fragmentation in multiples of the oligonucleosome length unit. The degree of DNA fragmentation increased when HHV-6-inoculated cells were cultured in the presence of TNF-alpha. Flow cytometry and Scatchard analysis of TNF receptors revealed an increase in the number of the p55 form of TNF receptors on JJHAN cells after HHV-6 inoculation. It also appeared that treatment with anti-Fas monoclonal antibody (MAb) induced marked apoptosis in HHV-6-inoculated cells. Transmission electron microscopy showed characteristics of apoptosis, such as chromatin condensation and fragmentation of nuclei, but virus particles were hardly detected in apoptotic cells. Two-color flow cytometric analysis using anti-HHV-6 MAb and propidium iodide revealed that DNA fragmentation was present predominantly in uninfected cells but not in productively HHV-6-infected cells. In addition, JJHAN cells incubated with UV light-irradiated and ultracentrifuged culture supernatant of HHV-6-infected cells appeared to undergo apoptosis. The present study demonstrated that both HHV-6 variants A and B induce apoptosis in CD4+ T cells by indirect mechanisms, as reported recently in human immunodeficiency virus type 1 infection.     

Cell-free synthesis of ornithine decarboxylase. Changes in mRNA activity in the liver of thioacetamide-treated rats

Ornithine decarboxylase (ODC)mRNA associated with free polysomes of rat liver was translated in a reticulocyte lysate cell-free system. Newly synthesized ODC protein was identified by specific immunoprecipitation, molecular size as determined by polyacrylamide gel electrophoresis with sodium dodecyl sulfate, and competition by excess unlabeled ODC in the immunoprecipitation. A single injection of thioacetamide was found to cause several fold increases in both immunotitratable ODC protein and polysomal ODC-mRNA activity, while it provoked a much larger increase in ODC activity in rat liver. The results indicate that the induction of hepatic ODC activity by thioacetamide treatment is due not only to an increase in the activity of polysomal ODC-mRNA but also to a translational and/or posttranslational control.     

Hormonal effects on the development changes of mouse small intestinal glycolipids

The composition of intestinal glycosphingolipids during normal and hormone-perturbed development was investigated. The concentrations of glycosphingolipids of mouse small intestine were affected by the injection of thyroxine or cortisone during suckling and weaning periods. GDla was reduced by the hormonal treatment among major gangliosides, GM3, GM1 and GD1a, of mouse small intestine during the suckling period. In contrast, asialo GM1 was precociously produced by the treatment, which scarcely found in control suckling mouse small intestine. The results showed that these hormones were related to developmental alteration of small-intestinal glycolipids.     

Antizyme to ornithine decarboxylase is present in the liver of starved rats.

Antizyme to ornithine decarboxylase (ODC) and ODC-antizyme complex were both present in liver cytosols of starved rats. The antizyme was identified by its molecular weight, kinetic properties, formation of a complex with ODC, and reversal of its inhibition by antizyme inhibitor. The average amount of antizyme in liver cytosols of starved rats was 0.1 unit/mg of protein, roughly corresponding to basal hepatic ODC activity in rats fed ad libitum. The presence of ODC-antizyme complex was detected by using antizyme inhibitor. These results indicate that antizyme participates in the regulation of ODC activity in vivo under physiological conditions.     

Stimulation of prostaglandin E2 synthesis by exogenous phospholipase A2 and C in rabbit kidney medulla slices.

We have investigated the effects of phospholipase A2 and C on the synthesis of prostaglandin E2 in rabbit kidney medulla and the release of fatty acids from the medulla slices. Exogenous phospholipase A2 [from Naja naja (Indian cobra) venom] and phospholipase C (from Clostridium welchii) stimulated prostaglandin E2 production in a dose-dependent manner. At the maximal effective concentrations (0.5 unit of phospholipase A2/ml, 2 units of phospholipase C/ml), phospholipase C increased prostaglandin E2 formation to the level observed with phospholipase A2. Phospholipase A2 enhanced the release only of unsaturated fatty acids, whereas phospholipase C stimulated the release of individual free fatty acids (C 16:0, C 18:0, C 18:1, C 18:2 and C 20:4). Moreover, p-bromophenacyl bromide inhibited phospholipase A2-stimulated prostaglandin E2 production and the release of fatty acids, but it had no influence on prostaglandin E2 formation and the release of fatty acids increased by phospholipase C, indicating that the stimulatory effect of phospholipase C is not mediated through the activation of endogenous phospholipase A2. These results suggest the presence of diacylglycerol lipase and monoacylglycerol lipase in the kidney and the importance of this pathway in prostaglandin synthesis by the kidney.