Developmental changes in gangliosides in cultured cerebellar granule neurons

The content and composition of gangliosides in cultures enriched in granule neurones and in astrocytes from rat cerebellum (P6–8) showed marked differences; astrocytes contained less than 10% of the amount of granule neurones and the profile was dominated by simple gangliosides with lactosyl ceramide backbone, while gangliosides of the b series, which constitute about 40% in nerve cells, were virtually undetectable. Granule cell maturation was accompanied by a 16-fold increase in the ganglioside content during the initial 8 days in a serum-supplemented medium (S⁺), reaching a plateau much earlier and at a higher level than observed in the cerebellum in vivo. Developmental changes were characterized, as in vivo, by a pronounced decrease in the GD3 proportion and an increase in the b series of gangliosides. Compared with S⁺, adhesion among cells and fibres is different in a serum-free medium (S^–) in which the rise in cellular ganglioside content was less (30%) but the developmental changes in ganglioside profile were similar. However, in cultures in S^– only, GM3 was not detectable, while the distribution of GM1 and GD3 indicated that maturation is retarded relative to cells in S⁺. Surface exposure of gangliosides (studied by the periodate/[³H]borohydride method) was similar under the two culture conditions. There was an initial delay, especially in S^–, in the insertion of gangliosides into the plasma membrane, while the labelling of GD3 (the dominant ganglioside of immature granule cells) was very low compared with all the other species throughout the whole cultivation time.Special issue dedicated to Dr. Frederick E. Samson.     

发酵法生产丁二酸的化学合成培养基的筛选

以产琥珀酸放线杆菌Actinobacillus succinogenes NJ113 为出发菌株,针对该菌株筛选出含有关键生长因子的化学合成培养基,其关键因子为谷氨酸（Glu）、蛋氨酸（Met）和生物素（VH）和烟酸（VPP）。结合原发酵培养基中的磷酸缓冲盐成分,最终得到的化学合成培养基配方（g/L）： CH3COONa 1.36,NaCl 1.0,MgCl2 0.2,CaCl2 0.2,Na2HPO4 0.31,NaH2PO4 1.6, KH2PO4 3,NH4HCO3 1.57,Glu 0.87,Met 0.11,VH 0.010,VPP 0.025。在3 L发酵罐上进行验证实验,50 g/L初始葡萄糖发酵70 h,丁二酸的质量浓度为45.2 g/L,丁二酸收率达到90.4%。与之前的半合成培养基发酵制备丁二酸相比,丁二酸的收率提高了25.2%,副产物也有很大幅度的减少。     

Biochemical and immunological characterization of exometabolites from an Indian strain of Leishmania donovani promastigotes grown in a chemically defined medium

Summary Exometabolites (EXOM) of an Indian strain of Leishmania donovani promastigotes isolated from a chemically defined medium by ultrafiltration consisted of proteins, glycoproteins, lipid and lipophosphopolysaccharide (LPPS). LPPS of Mr 40-28 kDa in SDS-PAGE could be labelled metabolically with [³²P]-phosphate and recovered in the aqueous phase of hot-phenol-water extraction of EXOM (PE-Aq) along with a glycoprotein of Mr 150-130 kDa (GP_150-130) . These two molecules could be eluted from DE-52 column with 200 mM NaCI (D₂). The 300 mM NaCl (D₃) and 400 mM NaCl (D4) eluates from DE-52 column contained one unsaturated polar lipid component. The LPPS had Rf value of 0.65–0.75 in Thin Layer Chromatography (TLC) using saturated phenol water solvent system. EXOM revealed 15 bands in SDS-PAGE of which proteins of Mr 84, 66, 56, 50 and 29 kDa were prominent. When EXOM were fractionated through Con A — Sepharose column, the fraction eluted with -methyl-D-mannoside (Con A-E) had seven bands as revealed by SDS-PAGE of which 25, 16, 13 and 12 kDa glycoproteins were prominent.The antigens present in EXOM can be classified as slower anodic migrating and faster anodic migrating antigens as revealed by immunoelectrophoresis (IEP). The slower anodic migrating antigens, LPPS and GP_150-130 recovered in PE-Aq and D₂ did not cross-react with kala- azar patients' sera but cross-reacted with homologous anti-promastigote sera. Two faster anodic migrating antigens which could be recovered in organic phase of hot phenol extraction of EXOM (PE-O) and eluted in D₃ and D₄ and Con A-E, cross-reacted with kala-azar patients' sera. The antigens of both the classes were sensitive to periodic acid oxidation.     

Culture of fetal alveolar epithelial type II cells in serum-free medium

Summary A serum-free culture medium (defined medium = DM) was elaborated by adding to Eagle’s minimum essential medium (MEM), non-essential amino acids, transferrin, putrescine, tripeptide glycyl-histidyl-lysine, somatostatin, sodium selenite, ethanolamine, phosphoethanolamine, sodium pyruvate, and metal trace elements. This medium was tested for its ability to support sustained surfactant biosynthesis in fetal alveolar epithelial type II cells. For up to 8 days, ultrastructure was maintained with persistance of lamellar inclusion bodies. Thymidine incorporation into DNA was enhanced about 50% in DM as compared with MEM, whereas it was enhanced 300% in 10% fetal bovine serum. With DM, the incorporation of tritiated choline into phosphatidylcholine (PC) of isolated surfactant material was about twice that with MEM. Deletion experiments evidenced the prominent role of pyruvate, transferrin, and selenium in the stimulation of surfactant PC biosynthesis. The addition of biotin to DM enhanced surfactant PC biosynthesis slightly and nonsurfactant PC biosynthesis markedly. The presence of nucleosides seemed unfavorable to the synthesis of surfactant PC. Type II cells responded to the addition of epidermal growth factor and insulinlike growth factor-I both by increased thymidine incorporation into DNA and choline incorporation into PC. It is concluded that DM represents a useful tool for cultivating type II cells without loss of their specialized properties and for studying the regulation of cell proliferation and surfactant biosynthesis in a controlled environment.     

Continuous multiplication of rabbit tracheal epithelial cells in a defined,hormone-supplemented medium

Summary An improved Ham’s F12 nutrient medium supplemented with epidermal growth factor (EGF), insulin (INS), and transferrin (TF) was developed for continuous proliferation and clonal growth of primary rabbit tracheal epithelial (TE) cells in culture. The addition of small quantities of fetal bovine serum (FBS) (0.01 to 0.1%) to cultures had little measurable stimulation on TE cell growth and plating efficiency. However, serum levels higher than 0.1% inhibited cell growth and also masked the growth stimulating activities of EGF and INS despite an increase in cell attachment. Under this defined, hormone-supplemented medium, and in the presence of a trace amount of serum (0.01%), 10 to 20% of the protease-dissociated TE cells attached to the culture dish followed by at least four population doublings during 7 to 10 d of culture. Clonal growth occurred at a seeding density of 17 cells/cm² with a plating efficiency of 6 to 8%. Confluent primary cultures could be passaged two to four times by treatment with a 0.1% trypsin-1 mM EDTA solution and a total of 10 to 30 population doublings of in vitro life span were obtained. The epithelial nature of cultured cells was confirmed by indirect immunofluorescent staining with antikeratin antibody as well as by transmission electron microscopy. This study shows that using this improved hormone-supplemented medium, rabbit TE cells can be maintained in culture for extended periods of time without the aid of a fibroblast feeder layer or explant tissue. This system could be useful for the study of cell differentiation of tracheal epithelium.     

Relative efficiency of selection methods to improve a ratio of two traits in Tribolium

Summary Three generations of upward selection for the egg mass/adult weight ratio were carried out in Tribolium castaneum. The experiment involved five lines: E — selected for increased egg mass; W — selected for decreased adult weight; R — selected for egg mass/adult weight directly; L — linear index selected with economic weights of m ₂: -m ₁ egg mass to adult weight (m ₁ and m ₂ are the means for adult weight and egg mass, respectively); NL — nonlinear index selected. Adult weight (7 days after adult emergence) and egg mass (between 7 and 11 days) were measured. The NL, E, and L lines had the greatest observed responses for the ratio; the R and W lines were not effective in improving the egg mass/adult weight ratio. It was expected that the NL line would be superior to the E, L, and R lines, and that the W line would respond the least. Observed response was significant for egg mass in the NL line, and for adult weight in the E, W, and R lines. Strong selection to increase egg mass seems to represent the optimal criterion for the ratio to be improved. The usefulness of nonlinear indices as selection criteria to improve a nonlinear trait, previously found to be optimal for a trait defined as the product of two component traits, appears to hold also for the selective improvement of the ratio of two traits. Serious limitations expected for direct selection of the ratio have been confirmed in this experiment.     

A Macromolecule-Free Partially Defined Medium for Trypanosoma cruzi*

SYNOPSIS. A macromolecule-free medium, containing in its defined part 3 salts, glucose, hemin, 21 amino acids, 3 lipids, and some undefined components obtained by dialysis of liver infusion, was developed for serial cultivation of Trypanosomacruzi at 28 C. The medium allows prolonged cultivation of T. cruzi by serial transfers and growth comparable to that obtained in more complex media, including those containing blood serum.     

Clonal growth of Chinese hamster cell lines in protein-free media

Summary A protein-free synthetic medium, MCDB 301, has been developed for clonal growth of Chinese hamster ovary cell lines. Medium F12 was developed originally for that purpose, but later failed to support good growth without small amounts of serum protein. Growth was restored by the addition of nonphysiological amounts of commercially prepared thyroxine or smaller amounts of the trace element selenium. The thyroxine preparation was shown to contain sufficient selenium to account for all of its growth-promoting activity. MCDB 301 contains increased concentrations of calcium chloride and glutamine, and a smaller amount of cysteine than medium F12. It also has been supplemented with 19 inorganic ions, in addition to selenium and those in medium F12, in order to insure against possible future deficiencies as chemicals are purified further. A Chinese hamster lung line which will not grow in MCDB 301 alone will grow when the medium is supplemented either with methylcellulose or with insulin. The growth-promoting activity is thought to be an impurity shared in common by both substances. The probable “essential” role of impurities in cellular growth in most synthetic media and the problems involved in attempting to develop a truly “defined” medium are discussed. This research was supported by Grant No. CA15305 from the National Cancer Institute.     

Survival,morphology, and catecholamine storage of chromaffin cells in serum-free culture: Evidence for a survival and differentiation promoting activity in medium conditioned by purified chromaffin cells

Adult bovine and young rat chromaffin cells cultured in serum-free medium were examined for their survival and differentiation following exposure to various additives, trophic agents and conditioned media. Adrenal chromaffin cells dissociated from 8 day old rats were maintained by dexamethasone, NGF and CNTF or without any additives in an N1-supplemented medium in similar numbers as in serum-containing medium for up to 6 days. Neuritic growth elicited by NGF or CNTF was enhanced in the absence of serum. Medium conditioned by purified bovine chromaffin cells improved cell survival and caused neurite outgrowth in a dose-dependent manner. The activiti(es) was sensitive to heat and trypsin and not blocked by the addition of anti-NGF antibodies. Bovine chromaffin cell survival was reduced by 30% when cells were maintained for one week in the absence as compared to the presence of serum. Addition of insulin, the N1 supplement, dexamethasone or dbcAMP single or in combinations improved the survival to different extents. A combination of insulin (5 g/ml) and dexamethasone (5×10^–6M) proved to be optimal in this respect. However, these supplements failed to restore the cellular catecholamine, noradrenaline and adrenaline contents to levels seen in the presence of serum. This was also true for a chromaffin cell-conditioned medium, which improved survival without elevating the catecholamine contents. Conditioned medium, however, partly restored a more physiological adrenaline-noradrenaline-ratio.Dedicated to Dr. E. M. Shooter and Dr. S. Varon as part of a special issue (Neurochemical Research, Vol. 12, No. 10, 1987).     

Growth and hepatospecific gene expression of human hepatoma cells in a defined medium

Summary The production of albumin, α-fetoprotein (AFP), and α-1 antitrypsin has been compared among human hepatoma cells cultured in medium containing serum, medium containing hormones and growth factors, and a basal medium containing selenium as the only supplement. Growth is sustained in all three media, and the expression of all three proteins was maintained for over 4 mo. in the various media. However, the quantitative production of albumin and AFP were dramatically different in the three media. Two hormones, insulin and triiodothyronine, influenced the level of secreted proteins. Triiodothyronine increases the amount of secreted albumin whereas insulin at 10 μg/ml reduced the level of total secreted protein.