The culture of rat myeloma and rat hybridoma cells in a protein-free medium

Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×10⁶ cells ml^–1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×10⁵ cells ml^–1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×10⁶ cells ml^–1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.Abbreviations F12 Hams F12 medium - DMEM Dulbeccos medium - RPMI RPMI 1640 medium - FBS foetal bovine serum     

Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture, clonal growth in low density culture, and stimulation to enter S phase in resting culture

A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin, transferrin, epidermal growth factor, poly-D-lysine, bovine albumin, oleic acid, and bovine alpha-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetal bovine serum (FBS), and colonies, albeit of smaller sizes, did form. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking alpha-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding alpha-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.     

Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture,clonal growth in low density culture,and stimulation to enter S phase in resting culture

Summary A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin transferrin epidermal growth factor, poly-d-lysine, bovine albumin, oleic acid, and bovine α-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetat bovine serum (FBS), and colonies, albeit of smaller sizes, diddform. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking α-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding α-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.     

Role of Bacillus amyloliquefaciens Y1 in the control of Fusarium wilt disease and growth promotion of tomato

The objective of this study was to examine the effects of Bacillus amyloliquefaciens Y1 on the control of Fusarium wilt disease and subsequent improvement in the growth of tomato plants. The Y1 strain strongly inhibited Fusarium oxysporum f. sp. lycopersici in vitro and also produced indole-3-acetic acid (IAA) in both the presence and absence of tryptophan. Over 96% of tomato seeds germinated when treated with either water, tryptone soy broth, or Y1 cultures, whereas root (5.40?cm) and shoot (5.15?cm) lengths were greatest in tomato seedlings treated with Y1 cultures that lacked tryptophan. Three experimental treatments – Black White medium (BW), BW medium with a commercial fungicide (BW?+?F), and Y1 culture inoculated in BW medium (Y1) – were applied to control Fusarium wilt disease under in vivo conditions. Application of Y1 culture and BW?+?F led to significantly lower disease incidence than did BW; moreover, shoot length and fresh and dry weight of both roots and shoots were greater in plants treated with Y1 than in plants treated with either BW or BW?+?F. A similar trend was observed for chitinase and β-1,3-glucanase activities in roots and leaves of tomato plants in all treatment groups over most of the experimental period. Finally, the presence of Y1 in the rhizospheric soils of Y1-treated plants resulted in a significant reduction in the populations of other bacteria. The results of our study demonstrated the effectiveness of Y1 not only in the control of Fusarium wilt disease but also for the enhancement of plant growth in cultivated tomato.     

Accumulation of iron by yersiniae.

Escherichia coli, Bacillus megaterium, and three species of yersiniae grew rapidly without significant production of soluble siderophores in a defined iron-sufficient medium (20 microM Fe3+). In iron-deficient medium (0.1 to 0.3 microM Fe3+) all organisms showed reduced growth, and there was extensive production of siderophores by E. coli and B. megaterium. Release of soluble siderophores by Yersinia pestis, Y. pseudotuberculosis, or Y. enterocolitica in this medium was not detected. Citrate (1 mM) inhibited growth of yersiniae in iron-deficient medium, indicating that the organisms lack an inducible Fe3+-citrate transport mechanism. Uptake of 59Fe3+ by all yersiniae was an energy-dependent saturable process, showing increased accumulation after adaptation to iron-deficient medium. Growth of Y. pseudotuberculosis and Y. enterocolitica but not Y. pestis on iron-limited solid medium was enhanced to varying degrees by exogenous siderophores (desferal, schizokinen, aerobactin, and enterochelin). Only hemin (0.1 pmol) or a combination of inorganic iron plus protoporphyrin IX promoted growth of Y. pestis on agar rendered highly iron deficient with egg white conalbumin (10 microM). Growth of Y. pseudotuberculosis and Y. enterocolitica was stimulated on this medium by Fe3+ or hemin. These results indicate that hemin can serve as a sole source of iron for yersiniae and that the organisms possess an efficient cell-bound transport system for Fe3+.     

Immunogenicity of Yersinia pestis grown on nutrient media at 28 and 37 degrees C

The immunogenicity of Y. pestis strain EV, grown in yeast-casein medium, yeast medium with Hottinger digest and yeast medium with sunflower-seed protein at 28 degrees C and 37 degrees C, for guinea pigs and white mice has been studied. As revealed in this study, these media ensure the formation of highly immunogenic populations of Y. pestis strain EV and, therefore, can be used for growing Y. pestis vaccine strains. Considerable fluctuations in the content of such highly protective antigen as fraction 1 do not affect the immunogenicity of live cultures of Y. pestis strain EV. This is due to the leveling of differences in the content of this antigen in the process of the multiplication of these bacteria in laboratory animals.     

Yarrowia lipolytica mutants devoid of pyruvate carboxylase activity show an unusual growth phenotype

We have cloned and characterized the gene PYC1, encoding the unique pyruvate carboxylase in the dimorphic yeast Yarrowia lipolytica. The protein putatively encoded by the cDNA has a length of 1,192 amino acids and shows around 70% identity with pyruvate carboxylases from other organisms. The corresponding genomic DNA possesses an intron of 269 bp located 133 bp downstream of the starting ATG. In the branch motif of the intron, the sequence CCCTAAC, not previously found at this place in spliceosomal introns of Y. lipolytica, was uncovered. Disruption of the PYC1 gene from Y. lipolytica did not abolish growth in glucose-ammonium medium, as is the case in other eukaryotic microorganisms. This unusual growth phenotype was due to an incomplete glucose repression of the function of the glyoxylate cycle, as shown by the lack of growth in that medium of double pyc1 icl1 mutants lacking both pyruvate carboxylase and isocitrate lyase activity. These mutants grew when glutamate, aspartate, or Casamino Acids were added to the glucose-ammonium medium. The cDNA from the Y. lipolytica PYC1 gene complemented the growth defect of a Saccharomyces cerevisiae pyc1 pyc2 mutant, but introduction of either the S. cerevisiae PYC1 or PYC2 gene into Y. lipolytica did not result in detectable pyruvate carboxylase activity or in growth on glucose-ammonium of a Y. lipolytica pyc1 icl1 double mutant.     

Levels of fraction I and VW antigens in cultures of the plague microbe grown on yeast-casein, yeast-Hottinger broth and yeast-sunflower oil cake media

The content of fraction 1 and VW-antigens in Y. pestis cultures grown in different media (yeast-casein medium, yeast medium with Hottinger digest, and yeast medium with sunflower-seed protein) was studied over the course of their growth by means of the antibody neutralization and microprecipitation in agar tests. The media under study were not inferior to the casein sulfuric hydrolysate-based medium used for control in their capacity for ensuring the synthesis of VW-antigens. The maximum accumulation of fraction 1 was observed in yeast medium with sunflower-seed protein. In all media the maximum content of fraction 1 was registered on day 3 of cultivation, and the maximum accumulation of VW-antigens on days 8-9 of incubation at 37 degrees C. The data obtained in this study make it possible to regard fraction 1 and VW-antigens as the secondary metabolites of Y. pestis.     

Freeze-fracture electron microscopic studies of age-related plasma membrane changes in Sporothrix schenckii

Characteristics of the plasma membrane of Sporothrix scheckii cells as revealed by freeze-fracture techniques have been classified into eight types (Y1, Y2a, Y2b, Y3a, Y3b, Y4a, Y4b, and Y5) in yeastlike cells grown under the following two conditions: brain heart infusion agar medium at 27 degrees C, and brain heart infusion agar medium at 37 degrees C. Type Y1 cells are yeastlike cells having smooth plasma membranes without any invagination. Typical characteristics of the other types are as follows: type Y2a, smooth plasma membranes with few trenchlike invaginations; type Y2b, wavy plasma membranes with few oval or irregularly formed invaginations; type Y3a, plasma membranes with many randomly distributed trenchlike invaginations; type Y3b, plasma membranes with many cocoonlike or irregularly formed invaginations; type Y4a, plasma membranes with longer trenchlike invaginations; type Y4b, plasma membranes with irregularly formed, enlarged invaginations; and type Y5, smooth or wavy plasma membranes with aggregations of intramembranous particles and with many vacuoles between cell walls and plasma membranes or in the cytoplasm in some cells. By counting the proportion of each type of yeastlike cell under the two conditions and with different cultivation periods, it appears that plasma membrane types change as aging progresses in the following order: type Y1, Y2a, Y3a, Y4a, and Y5 in conidia and type Y1, Y2b, Y3b, Y4b, and Y5 in yeastlike vegetative cells. These observations provide us with an important advantage when studying the effects of antifungal agents on the plasma membrane of Sporothrix scheckii, as it is important to know the natural course of changes in membrane structure during aging.     

Molar Growth Yield of Streptococcus faecalis on Pyruvate

Y(pyruvate) was 17.3, similar to Y(arginine), for Streptococcus faecalis 6783 grown statically in a complex medium in 1 atm of air.     

Effects of an inhibitor of glucosylceramide synthase on glycosphingolipid synthesis and neurite outgrowth in murine neuroblastoma cell lines.

1-Phenyl-2-decanolyamino-3-morpholino-1-propanol (PDMP), an effective inhibitor of UDP-glucose:ceramide glucosyltransferase, caused inhibition of cell growth in murine neuroblastoma cell lines. Metabolic labeling of glycosphingolipids with [14C]galactose in NS-20Y, Neuro2a, and N1E-115 cells showed reduced incorporation of radioactivity into gangliosides and neutral glycosphingolipids when threo-PDMP was present in the medium. Treatment of NS-20Y cells with threo-PDMP resulted in a time-dependent decrease in mass levels of gangliosides and neutral glycosphingolipids. After 24 h in the presence of 50 microM threo-PDMP, neutral glycosphingolipid mass was reduced to 32%, where glucosylceramide was the most affected (90% decrease). The ganglioside mass was reduced to 57% of the original content. Neurite outgrowth from neuroblastoma cells in serum-free medium was significantly inhibited by threo-PDMP in a dose-dependent manner. Threo-PDMP also caused retraction of neurites which had been induced to extend in serum-free medium. Pretreatment of cells with GM1 partially restored the ability of NS-20Y cells for neurite outgrowth in the medium containing threo-PDMP. These results suggest a possible role for glycosphingolipids in neurite outgrowth of murine neuroblastoma cells.     

木屑马铃薯培养基促进香菇单核体菌丝体生长的表达谱分析

香菇单核体菌株在传统PDA培养基上生长时具有生长缓慢、容易老化等问题,本研究以1株香菇双核体Y0040以及相对应的2株单核体(Y0040-1和Y0040-3)为研究材料,通过添加不同比例木屑粉的PDA培养基筛选适合香菇单核体生长的配比,结果表明添加木屑能够显著促进单核体菌丝的生长,最适添加比例为2%。将Y0040-1和Y0040-3在PDA和2%木屑PDA上培养后进行转录组表达谱差异分析,结果显示Y0040-1和Y0040-3两个单核菌株在木屑-PDA培养基上生长有1066个共有的差异表达基因,进一步对其注释发现,这些差异基因在细胞结构合成以及碳水化合物代谢等途径上得到富集。同时1066个共有的差异基因中有113个共上调,富集于氧化还原反应,267个共下调主要富集于蛋白质折叠和去折叠等途径。进一步对1066个差异基因进行CAZYmes家族和木质纤维素酶分析,发现有36个家族基因差异表达,包括了4个多铜氧化酶、6个β-葡萄糖苷酶和2个内β-1,4-葡聚糖酶,其中多铜氧化酶基因表达在木屑培养基上都显著上升。木质纤维素降解酶基于氧化还原反应等将木质素降解为菌丝体生长发育所必需的小分子单糖,可...     

海洋红酵母Y2发酵产类胡萝卜素条件的研究

目的 对海洋红酵母Y2高产类胡萝卜素的发酵条件进行优化.方法 在摇瓶条件下,研究培养基成分和培养条件对海洋红酵母Y2生长和类胡萝卜素合成的影响,同时进行海洋红酵母Y2发酵过程的动态分析.结果 海洋红酵母Y2优化培养基组合为葡萄糖45 g/L,蔗糖15 g/L,酵母粉5 g/L,蛋白胨2.5 g/L,磷酸二氢钾1 g/L,磷酸二氢钠3 g/L,硫酸镁7.5 g/L,氯化钾3 g/L,氯化钠5 g/L.最适培养参数为:温度20℃,培养基初始pH为5,接种量为10％,250 mL摇瓶装液量为10～50 mL.类胡萝卜素的合成主要集中在对数生长期和稳定期.海洋红酵母Y2最适收获时间为72 h.种龄以36 h为宜.结论 利用优化培养基,在最适条件下培养海洋红酵母Y2,类胡萝卜素产量达到4.97 mg/L,比基础培养基提高了60.32％.     

Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells

We characterized the expression and functional properties of the ADP-sensitive P2Y(1) and P2Y(12) nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long-term serum starvation, cell morphology changed from fibroblast-like flat to round, the adhesion pattern changed, cell-cycle arrest was induced, extracellular signal-regulated kinase (ERK1/2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y(12) receptor relative to P2Y(1) was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y(1) receptor was low, and the P2Y(12) receptor was the major player, responsible for ADP-evoked signal transduction. The P2Y(12) receptor activated ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR-C69931MX, a specific antagonist of the P2Y(12) receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y(1) receptor, indicating the inhibitory role of P2Y(1) in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y(1) to P2Y(12) would appear to be a new and important self-regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation.     

Arginine vasopressin inhibition of cyclin D1 gene expression blocks the cell cycle and cell proliferation in the mouse Y1 adrenocortical tumor cell line

Arginine vasopressin (AVP) is a nonapeptide long known as an endocrine and paracrine regulator of important systemic functions, namely, vasoconstriction, gluconeogenesis, corticosteroidogenesis, and excretion of water and urea. Here we report, for the first time, that AVP specifically inhibits expression of the cyclin D1 gene, leading to cell cycle blockage and halting cell proliferation. In G0/G1-arrested mouse Y1 adrenocortical tumor cells, maintained in serum-free medium (SFM), AVP mimics FGF2, promoting rapid ERK1/2 activation (5 min) followed by c-Fos protein induction (2 h). PKC inhibitor Go6983 and PI3K inhibitors wortmannin and LY294002 all inhibit ERK1/2 activation by AVP, but not by FGF2. Thus, AVP and FGF2 concur to activate ERK1/2 by different regulatory pathways. However, AVP is not a mitogenic factor for Y1 cells. On the contrary, AVP strongly antagonizes FGF2 late induction (2-5 h) of the cyclin D1 gene, down-regulating both cyclin D1 mRNA and protein. AVP inhibition of cyclin D1 expression is sufficient to block G1 phase progression and cell entry into the S phase, monitored by BrdU nuclear labeling. In addition, AVP completely inhibits proliferation of Y1 cells in 10% fetal calf serum (10% FCS) medium. On the other hand, ectopic expression of the cyclin D1 protein renders Y1 cells resistant to AVP for both entry into the S phase in SFM and continuous proliferation in 10% FCS medium. In conclusion, inhibition of cyclin D1 expression by AVP is an efficient mechanism of cell cycle blockage and consequent proliferation inhibition in Y1 adrenocortical cells.     

ROCK抑制剂Y27632对小胶质细胞炎性分泌的影响

目的观察ROCK抑制剂Y27632对小胶质细胞活化及其炎性分泌的作用。方法采用小胶质细胞系BV2细胞复苏后传代培养,分为对照组和Y27632干预组;在0、3h、6h、18h和24h收取细胞和细胞培养基;采用免疫荧光细胞染色测定各组BV2细胞上Ibal的表达,ELISA法测定各组培养基中IL-1B和TNF-α的浓度变化。结果Y27632可明显改变BV2细胞形态,干预组细胞比对照组细胞的胞体更大,细胞突起增多增长,细胞形态改变在干预6h时最为显著。与对照组相比,Y27632干预后BV2细胞分泌的IL-1β和TNF-α降低,在18h时抑制效果最为显著（P〈0．01）。结论ROCK抑制剂Y27632可诱导BV2细胞活化及炎性分泌降低,提示Rho／ROCK信号通路在小胶质细胞活化及炎性因子分泌中发挥了重要作用。     

A new chromogenic agar medium for detection of potentially virulent Yersinia enterocolitica

Several outbreaks of foodborne yersiniosis have been documented and this disease continues to be source of infections transmitted through foods. The selective agars most commonly used to isolate Yersinia enterocolitica in clinical, food and environmental samples, cefsulodin-irgasan-novobiocin (CIN) and MacConkey (MAC) agars, lack the ability to differentiate potentially virulent Y. enterocolitica from other Yersinia that may be present as well as some other bacterial spp. This study proposes the use of an agar medium, Y. enterocolitica chromogenic medium (YeCM), for isolation of potentially virulent Y. enterocolitica. This agar contains cellobiose as the fermentable sugar, a chromogenic substrate and selective inhibitors for suppression of colony formation by many competing bacteria. All strains of potentially virulent Yersinia of biotypes 1B, and biotypes 2-5 formed convex, red bulls-eye colonies on YeCM that were very similar to those described for CIN agar. However, Y. enterocolitica biotype 1A and other related Yersinia formed colonies that were purple/blue on YeCM while they formed typical red bulls-eye colonies on CIN agar. When a mixture of potentially virulent Y. enterocolitica biotype 1B, Y. enterocolitica biotype 1A and 5 other bacterial species was used to artificially contaminate tofu and then spread-plated on three selective agars, Y. enterocolitica biotype 1B colonies were easily distinguished from other strains on YeCM. However, Y. enterocolitica biotype 1B colonies were indistinguishable from many other colonies on CIN and only distinguishable from those of C. freundii on MAC. When colonies were picked and identified from these agars, typical colonies from YeCM were confirmed only as Y. enterocolitica biotype 1B. Typical colonies on CIN and MAC were found to belong to several competing species and biotypes.     

NMR based metabonomics study of NPY Y5 receptor activation in BT-549, a human breast carcinoma cell line

Overexpression of neuropeptide Y (NPY) and its receptors has been found in various cancers. In our previous study, we demonstrated expression of NPY Y5 receptor (Y5R) in various breast cancer cell lines along with Y1 receptor. In Y5R expressing BT-549 cells, NPY induced cell proliferation that was blocked by Y5R-selective antagonist CGP1683A (CGP). Here, NMR-based metabonomics was used to monitor the metabolic profile of BT-549 cells in the presence of NPY and CGP to assess the effect of Y5R activation and inhibition during NPY-induced cell proliferation. To study changes in intra and extra cellular metabolites in response to various treatments, 1D ¹H-NMR spectra of both hydrophilic cell extracts and growth medium were recorded from BT-549 with three treatments: (1) NPY, (2) CGP, and (3) CGP followed by NPY (CGP/NPY). Principal component analysis and statistical significance analysis indicated changes in intracellular concentrations of seven metabolites in hydrophilic cell extracts with NPY treatment: decreases in lactate, succinate, myo-inositol, and creatine, and increases in acetate, glutamate, and aspartate. A significant increase in intracellular lactate level and attenuation of other metabolites to baseline was detected in CGP/NPY group. Also, significant decreases in lactate and increases in pyruvate were observed in growth medium from NPY treated cells. Based on the metabonomics analysis, Y5R activation induces cell proliferation by increasing the rate of glycolysis, glutaminolysis, and TCA cycle. Inhibition of Y5R by CGP counteracts NPY-induced changes in cellular metabolites. These changes may play a role in cell proliferation and migration by NPY through Y5R activation.