An envelope-shaped film culture vessel for plant cell suspension cultures and metabolite production without agitation

An envelope-shaped film culture vessel (named Culture Bag) made of fluorocarbon polymer film, which is much more permeable to oxygen, nitrogen and carbon dioxide than other films, was found to be suitable to grow plant cells in liquid medium without agitation. Proliferous BY-2 tobacco cells showed almost the same growth in a Culture Bag of 12.5 m-thick film as that in a shake flask; the growth was lower in a Culture Bag of a thicker film. Lithospermum erythrorhizon cells produced almost the same amount of red naphthoquinone pigments (shikonin derivatives) in a Culture Bag of 12.5 m-thick film as those in a shake flask although the productivity was suppressed as the film thickness increased. L. erythrorhizon cells in a Culture Bag produced much less abnormal stress metabolites (orange-colored benzoquinone derivatives) than those in a shake flask, suggesting that culturing cells in the Culture Bag was less stressful due to its stationary liquid environment.     

Effect of platelet-derived growth factor on DNA synthesis and gene expression in bone marrow stromal cells derived from adult and old rats

The effects of platelet-derived growth factor (PDGF) on DNA synthesis and mRNA expression of osteoblast markers in marrow stromal cells derived from adult (6 months) and old (24 months) rats were examined. Treatment of stromal cells from adult rats with dexamethasone induced the appearance of osteoblast-like cells. PDGF partially also inhibited the differentiation of stromal cells induced by dexamethasone. In cultures of serum-starved stromal cells, PDGF stimulated [³H]-thymidine incorporation into DNA in a dose-dependent manner with a maximum stimulation of 15-fold at 500 ng/ml. By comparison, insulin-like growth factor (IGF-I) has a small effect on [³H] -thymidine incorporation. The effect of PDGF and IGF-I on DNA synthesis was additive. Treatment of the confluent stromal cells from adult rats with PDGF increased the mRNA level of osteopontin fourfold without any significant effect on alkaline phosphatase and type I collagen mRNAs. In contrast, dexamethasone stimulated the mRNA expression of alkaline phosphatase, type I collagen, and osteopontin 2.1-, 2.3-, and 14-fold, respectively. Addition of PDGF to dexamethasone-treated cells failed to induce any further increase in osteopontin expression whereas the expression of alkaline phosphatase and type I collagen was partially reduced. The expression of osteocalcin mRNA was negligible in stromal cells but stimulated several fold by dexamethasone and 1,25(OH)₂D₃. PDGF inhibited drastically the elevation of osteocalcin mRNA. In contrast, IGF-I stimulated type I collagen expression 100% without any appreciable effect on the expression of osteopontin and alkaline phosphatase. The stimulatory effect of PDGF on osteopontin expression was augmented by IGF-I. Furthermore, PDGF attenuated the stimulatory effect of IGF-I on type I collagen expression. The responses of cultured cells from old rats to growth factors were also examined. PDGF or PDGF plus IGF-I increased [³H]-thymidine incorporation in stromal cells from old rats but to a lesser extent. However, PDGF was equally effective in stimulating osteopontin expression in cells from both adult and old rats. We concluded that PDGF is a potent mitogen but that the response of stromal cells from old rats is impaired. In addition, PDGF stimulates osteopontin expression in stromal cells and this effect is not age dependent. © 1995 Wiley-Liss, Inc.     

Lipopolysaccharide inhibits activation of latent transforming growth factor-β in bovine endothelial cells

The activation of latent transforming growth factor-β (TGF-β) by vascular endothelial cells (ECs) is regulated by cellular plasminogen activator (PA)/plasmin, transglutaminase (TGase), and latent TGF-β levels. Because lipopolysaccharide (LPS) has been reported to reduce EC surface plasmin levels by increasing the production of the inhibitor of PA, PA inhibitor-1 (PAI-1), we have tested whether LPS might suppress latent TGF-β activation in ECs using two different systems, namely, bovine aortic ECs (BAECs) cocultured with smooth muscle cells (SMCs) and BAECs treated with retinol. BAECs were either cocultured with SMCs after treatment with 15 ng/ml LPS or were treated with 2 μM retinol and/or 10 ng/ml LPS, and the expression of PA, surface plasmin, TGase, and the amounts of active and latent TGF-β secreted into the culture modium were measured. The downregulation of surface PA/plasmin levels with LPS was accompanied by a profound decline of both TGase and latent TGF-β expression as well as the suppression of surface activation of latent TGF-β. The effect was dependent on the concentration of LPS and on treatment time. The formation of TGF-β did not occur in cells maintained in LPS-contaminated culture medium. © 1995 Wiley-Liss, Inc.     

Possible involvement of ethylene-activated {beta}-cyanoalanine synthase in the regulation of cocklebur seed germination

A possible involvement of ß-cyanoalanine synthase(CAS: EC 4.4.1.9 [EC] ) in germination processes of seeds was demonstratedusing pre-soaked upper seeds of cocklebur (Xanthium pennsylvanicumWallr.). Pretreatment in anoxia not only with KCN but also cysteine,as the substrates for CAS, stimulated the subsequent germinationof cocklebur seeds in air. However, the effect of cysteine wasmanifested even in air when applied together with C₂H₄, andits effect was further enhanced in combination with KCN. Thegermination-stimulating effect of KCN was intensified by C₂H₄only when 0₂ was present. In contrast, serine, another substrateof CAS, was effective in air only when combined with C₂H₄ and/orKCN. The addition of cysteine greatly reduced the cyanogenicglycoside content of seeds, but increased HCN evolution. Onthe other hand, glutathione did not have any effect on cockleburseed germination, HCN evolution or bound cyanogen content, suggestingthat cysteine is not acting as a reducing reagent. It is suggestedthat CAS regulates the process of cocklebur seed germinationby the dual action of enlarging the pool of amino acids andsupplying sulphydryl bases, the latter being more determinatelyimportant. Serine is effective only via the former action, whilecysteine would act via both. Key words: Cyanide, cyanogenic glycoside, ß-cyanoalanine synthase, seed germination, Xanthium pennsylvanicum     

Expression of the T antigen on a T-lymphoid cell line, SupT1

We have measured glycosyltransferase activities of SupT1 cells, a T-lymphoid cell line shown to react with autoantibodies in the sera of many HIV patients. Since considerable -N-acetylgalactosaminyl-transferase and 1, 3 galactosyl-transferase activities were found in SupT1 cells, at least the O-glycan core 1 structure can probably be synthesized. FACS analysis using an anti-T monoclonal antibody showed expression of the T antigen (Gal 1-3 GalNAc). Glycoproteins with the T antigen were isolated by immunoprecipitation with the anti-T antibody from a SupT1 cell lysate labelled metabolically with³H-glucosamine and then analysed by SDS-PAGE. It was revealed that the precipitate contained a glycoprotein with a molecular weight corresponding to that of leukosialin. O-glycans were prepared from the immunoprecipitate by alkaline-borohydride treatment and then fractionated on Bio-Gel P-2, GalNAcOH and Gal-GalNAcOH being identifiedinter alia. These results suggest that an anti-T antibody may be included in the autoantibodies found in HIV-1 infected individuals.     

Cloning and expression of a porcine UDP-GalNAc: polypeptideN-acetylgalactosaminyl transferase

By employing a bovine UDP-N-acetylgalactosamine: polypeptideN-acetylgalactosaminyl transferase (O-GalNAc transferase) cDNA as a probe, we isolated four overlapping cDNAs from a porcine lung cDNA library. Both the nucleotide sequence of the porcine cDNA and the predicted primary structure of the protein (559 amino acids) proved to be very similar to those of the bovine enzyme (95% and 99% identity, respectively). Transient expression of the clone in COS-7 cells, followed by enzymatic activity assays, demonstrated that this cDNA sequence encodes a porcine O-GalNAc transferase. The intracellular O-GalNAc transferase activity was increased approximately 100-fold by transfecting cells with the porcine cDNA.Abbreviations O-GalNAc transferase UDP-N-acetylgalactosamine: polypeptideN-acetylgalactosaminyltransferase - PCR polymerase chain reaction - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - GnT-III UDP-N-acetylglucosamine: -mannoside -1,4N-acetylglucosaminyltransferase III     

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.     

Over-production of Thermus protease in dense culture of Escherichia coli using two carbon sources

Escherichia coli JM109(DE3) harboring expression plasmid pkAQNÆC30, which carries the Thermus protease aqualysin I (AQI) gene, was cultivated with glucose as a sole carbon source. The final cell concentration was over 15 g dry weight/l and the amount of AQI produced reached approximately 130 kU/ml broth. Moreover, by using two carbon sources, glucose and glycerol, the production yield was increased to over 200 kU AQI/ml, while suppressing the formation of inhibitory acetic acid.     

Unique tissue distribution of a mouse macrophage C-type lectin

We examined mouse tissue for the expression of macrophage galactose/N-acetylgalactosamine-specificC-type lectin using a rat monoclonal antibody (mAb) specificfor this lectin (mAb LOM-14). The binding of mAb LOM-14 wasdetected in detergent extracts from tissue by means of immunoblottinganalysis. It was shown that this mAb did not cross-react withmouse hepatic lectins, a structural homologue. The macrophagelectin was widely distributed among various mouse tissues asjudged by the affinity isolation followed by the immunochemicaldetection. The exceptions were brain, liver, kidney, small intestine,and peripheral blood. Extracts from these organs exhibited,at best, very weak signals upon mAb LOM-14 binding, despitethe presence of cells expressing macrophage markers. The mostintense signal was observed in the extract from skin, suggestingthat cells expressing this lectin are abundant in skin. Thetissues shown to contain this lectin were further investigatedby immunohistochemical staining of the sections. Cells weredistributed in the connective tissue and in the interstice,particularly the dermis and subcutaneous layer of skin. Cellslocalized in the epithelium of skin (epidermis) or other epitheliathat we examined were not stained. Perivascular localizationof cells stained with mAb LOM-14 was also demonstrated in cardiacand skeletal muscle tissues. Immunoelectron microscopy revealedthe presence of this lectin along the rough endoplasmic reticulum.In conclusion, the distribution of C-type lectin specific forgalactose/N-acetylgalactosamine in mice was unique. The connectivetissue-specific distribution should provide important informationon the biological role of this lectin. lectin macrophage calcium-type lectin connective tissue     

Physical effects of negative air ions in a wet sauna

 The physical effects of negative air ions on humans were determined in an experimental sauna room equipped with an ionizer. Thirteen healthy persons took a wet sauna bath (dry bulb temperature 42° C, relative humidity 100%, 10 min exposure) with or without negative air ions. The subjects were not told when they were being exposed to negative air ions. There were no differences in the moods of these persons or changes in their blood pressures between the two saunas. The surface temperatures of the foreheads, hands, and legs in the sauna with negative ions were significantly higher than those in the sauna without ions. The pulse rates and sweat produced in the sauna with ions were singificantly higher than those in the sauna without ions. The results suggest that negative ions may amplify the effects on humans of the sauna. Received: 31 March 1995 / Revised: 25 July 1995 / Accepted: 26 July 1996