The limitation of growth of Trypanosoma musculi in vitro

Trypanosoma musculi grow readily in vitro provided their growth is supported by mammalian cells. In the presence of murine spleen cells, or spleen cell-conditioned medium, the parasites increase by 100-fold, or more, in a period of 5–6 days. Growth ceases abruptly and death of the parasites soon follows. The reason for the termination of growth has been obscure and is the subject of this report. Termination of growth is not due to an immunological process; not even of ablastin affecting epimastigote reproduction. Instead it appears that other growth inhibitory substances are responsible. Culture medium, collected from spent cultures on day 8 after initiation, inhibits T. musculi growth in fresh medium in dose-dependent fashion. No inhibitory substances were present in medium collected earlier, during the phase of rapid parasite growth. These inhibitory substances appeared to be derived from the parasites rather than the cocultivated spleen cells.     

Serum- and animal tissue-free medium for transport and growth of Helicobacter pylori

The important human gastric pathogen Helicobacter pylori is the subject of many studies, and as a consequence it is frequently being transported between national and international laboratories. Unfortunately, common bacterial growth and transport media contain serum- and animal tissue-derived materials, which carry the risk of spreading infectious diseases. We have therefore developed a growth and transport medium for H. pylori, designated 'Serum- and Animal Tissue-Free Medium' (SATFM), which does not contain serum- or animal tissue-derived components. SATFM supported growth of H. pylori isolates to similar levels as obtained with serum-supplemented Brucella medium, and SATFM with 0.5% agar supported transport and storage of H. pylori strains, as 4/4 reference strains and 11/11 clinical isolates survived for at least 3 days at room temperature in SATFM, with some strains (2/15) even surviving for up to 7 days. In conclusion, SATFM can be used both as transport and growth medium for H. pylori. The formulation of SATFM may allow its use in international transport of H. pylori, and may also allow certified use in immunization studies requiring growth of H. pylori and other bacterial pathogens.     

A comparison of chromosomal radiosensitivities of somatic cells of mouse and man.

A cell culture technique for quantitative analysis of radiation-induced chromosome aberrations in somatic cells has been developed and used for the comparison of chromosomal sensitivity of skin cells of mouse and man to 60Co-gamma-rays. This includes culture of irradiated tissues or cells in culture in arginine and isoleucine-deficient medium and subsequent refeeding with complete medium (CM). With this technique, radiation-induced chromosome aberrations can be analyzed selectively in the cells exposed in G1 phase and recovered at their first post-irradiation mitosis. When tested on the human embryonic cells, the dicentric yield was essentially the same whether they were skin cells irradiated in silu or cultured cells at various in vitro passages irradiated in vitro. In contrast, when studied in the skin cells irradiated in silu, mouse embryos and newborns were insensitive to the induction of dicentrics. In young mice on day II however, the sensitivity was at a level comparable to that in human embryonic cells and it was intermediate on day 4. Such embryonic insensitivity of the mouse cells was rapidly lost during serial transfer in vitro; and, when tested at 4th or later subculture generations, mouse and human cells were equally sensitive to the induction of dicentrics. These results suggest that the chromosomal radiosensitivity is essentially the same for mouse and human cells but can be modified by some biological factors, possibly DNA repair mechanisms, which differ between species as well as among the states of differentiation of particular cell types. Special attention was paid to the parellelism between the age-dependent changes in the chromosomal, mutational and carcinogenic radiosensitivities in the mouse. If this parallelism can be carried over to man, human pre-natal irradiation will not present any reduced genetic hazards.     

Growth of human tumor cell lines in transferrin-free,low-iron medium

Summary Iron is essential for tumor cell growth. Previous studies have demonstrated that apart from transferrin-bound iron uptake, mammalian cells also possess a transport system capable of efficiently obtaining iron from small molecular weight iron chelates (Sturrock et al., 1990). In the present study, we have examined the ability of tumor cells to grow in the presence of low molecular weight iron chelates of citrate. In chemically defined serum-free medium, most human tumor cell lines required either transferrin (5 μg/ml) or a higher concentration of ferric citrate (500 μM) as an iron source. However, we have also found that from 13 human cell lines tested, 4 were capable of long-term growth in transferrin-free medium with a substantially lower concentration of ferric citrate (5 μM). When grown in medium containing transferrin, both regular and low-iron dependent cell lines use transferrin-bound iron. Growth of both cell types in transferrin medium was inhibited to a certain degree by monoclonal antibody 42/6, which specifically blocks the binding of transferrin to the transferrin receptor. On the contrary, growth of low-iron dependent cell lines in transferrin-free, low-iron medium (5 μM ferric citrate) could not be inhibited by monoclonal antibody 42/6. Furthermore, no autocrine production of transferrin was observed. Low-iron dependent cell lines still remain sensitive to iron depletion as the iron(III) chelator, desferrioxamine, inhibited their growth. We conclude that low-iron dependent tumor cells in transferrin-free, low-iron medium may employ a previously unknown mechanism for uptake of non-transferrin-bound iron that allows them to efficiently use low concentrations of ferric citrate as an iron source. The results are discussed in the context of alternative iron uptake mechanisms to the well-characterized receptor-mediated endocytosis process.     

A neutron small-angle scattering study of bovine fibrinogen.

A number of glycyl-tRNA synthetase (glyS) mutants have been isolated as glycine auxotrophs in Salmonella typhimurium. One of the mutants, glyS141, has a glycyl-tRNA synthetase with a K_m for glycine that is 700 times higher than the wild-typeK_m. Prototrophic revertants glyS141 occur at high spontaneous frequencies (>5 × 10^?5). The majority of these revertants contain large tandem duplications including the mutant glyS gene. Some of the duplications cover at least 22% of the chromosome. The duplications overlap with a large duplication isolated previously by a different selection procedure (Straus &; Hoffmann, 1975). Evidence has been obtained which suggests that formation of the duplications may occur by recA-dependent recombination. The Gly⁺ phenotype of revertants carrying the duplications does not appear to be explainable simply by the increased gene dosage of glyS.     

The use of psyllium (isubgol) as an alternative gelling agent for microbial culture media

Psyllium (Isubgol), the mucilage husk of Plantago ovata, was successfully used as an alternative gelling agent (5% w/v as ground husk for pouring medium and 4% w/v as ground husk in combination with 0.5% w/v agar for slant) for microbial culture. Most of the undesirable features of psyllium-gelled media (slant as well as pouring) were removed by adding the u.v.-treated (20min) or oven-sterilized (120^°C for 1h) psyllium as ground husk to autoclaved medium at 50–60^°C under aseptic condition just before pouring.     

Media formulation options and manufacturing process controls to safeguard against introduction of animal origin contaminants in animal cell culture

Technical limitations and evolution of therapeuticapplications for cell culture-derived products haveaccelerated elimination of animal-derived constituentsto minimize inadvertent introduction of adventitiousviral or prion agents. Practical considerationsdemand adequate emphasis both on design of theserum-free/protein-free culture environment and onnutrient media manufacturing process controls. Protein components may be acceptable, given adequateattention to synthetic process, sourcing (e.g.,geographic location and endemicity, species andtissue/organ) and validated treatment method. Variousoptions exist for re-engineering of traditionalserum-free formulations (containing insulin,transferrin and other protein factors) withnon-protein substitutes. Caution must also beexercised with sourcing of non-protein additives,particularly amino acids and lipids, to avoidintroducing adventitious contaminants. Simpleguidelines facilitate adaptation, cryopreservation andrecovery of many cell types within a protein-freeculture environment. Scrupulous maintenance offacility and equipment and monitoring of processwater, air handling systems and technical personnelare required to ensure that approved raw materials arecorrectly formulated and dispensed. Validatedsanitization processes provide additional assuranceagainst cross-contamination from previous batches ina multi-use facility.     

A BROAD SPECTRUM ARTIFICIAL SEA WATER MEDIUM FOR COASTAL AND OPEN OCEAN PHYTOPLANKTON1

A new artificial seawater medium has been tested with 83 strains of coastal and open ocean phytoplankton from 11 different algal classes. The cultures were carried through four transfers, representing a period of eight weeks for most species. Only three species could not be maintained in the enriched artificial seawater, and 16 species, mainly from the Prymnesiophyceae and Dinophyceae, had reduced final cell yields compared to those grown in enriched natural seawater. Since 77% of the species tested grew equally well in enriched artificial or natural seawater and more than 95% could be maintained in the artificial medium, this recipe is useful over a broad spectrum of species. The artificial seawater base was enriched with a modified ES enrichment solution; the primary modifications were the omission of Tris and the addition of Si. Enriched medium was autoclaved without precipitation by lowering the pH before autoclaving. This was accomplished by adding equimolar amounts of Na-HCO₃ and HCl which produced NaCl and CO₂ during the heating process. When no pH buffer was used, precipitation could only be avoided by autoclaving the artificial seawater base as two separate salt solutions (with Ca and Sr separated from CO₃^?2 and SO₄^?2), cooling, mixing and aseptically adding the sterilized enrichment solution.     

Rapid Growth of Acanthamoeba In Defined Media; Induction of Encystment By Glucose-Acetate Starvation

Defined media are described that support 14-20 h generation times for Acanthamoeba castellanii and A. rhysodes in monolayer cultures. the media differ in minor ways from previously described media, but the growth rates are greatly improved over previously reported values. Maximum growth rates were observed for A, castellanii in a complex medium containing 21 amino acids, but near-maximum rates could be achieved in relatively simple media containing 9 amino acids. Growth occurred with 6 amino acids, as reported by others, but generation times exceeded 30 h. Amitosis was a common problem during early subcultures in defined media, but became infrequent after repeated transfers. Synchronous encystment resulting in 70-80% cyst formation could be induced in the defined media by glucose and acetate starvation. the rate of encystment varied with cell density at the time of starvation and was optimal at initial densities of 400-800 amebae/mm2.     

Waste molasses alone displaces glucose-based medium for microalgal fermentation towards cost-saving biodiesel production

The by-product of sugar refinery—waste molasses was explored as alternative to glucose-based medium of Chlorella protothecoides in this study. Enzymatic hydrolysis is required for waste molasses suitable for algal growth. Waste molasses hydrolysate was confirmed as a sole source of full nutrients to totally replace glucose-based medium in support of rapid growth and high oil yield from algae. Under optimized conditions, the maximum algal cell density, oil content, and oil yield were respectively 70.9 g/L, 57.6%, and 40.8 g/L. The scalability of the waste molasses-fed algal system was confirmed from 0.5 L flasks to 5 L fermenters. The quality of biodiesel from waste molasses-fed algae was probably comparable to that from glucose-fed ones. Economic analysis indicated the cost of oil production from waste molasses-fed algae reduced by 50%. Significant cost reduction of algal biodiesel production through fermentation engineering based on the approach is expected.     

Effects of proliferation,maturation, and desiccation methods on conversion of soybean somatic embryos

Summary Cermination of soybean [Glycine max (L.) Merrill] somatic embryos and conversion to whole plants are generally low. This study was conducted to investigate the effects of proliferation, maturation, and desiccation methods on conversion of soybean somatic embryos to plants. Soybean cv. Jack somatic embryos, proliferated on a solid medium containing 90.5 μM (20 mgl⁻¹) 2.4-dichlorophenoxyacetic acid (2.4-D) (MSD20), showed a regeneration rate signficantly higher than those proliferated in a liquid medium containing 45.25 μM (10mgl⁻¹) 2,4-D (FN Lite). When a liquid medium without 2,4-D and B5 vitamins (FN Superlite) was used for maturation, the duration of time necessary for embryo development could be shortened by more than a month compared to maturation on a standard solid medium (MSM6AC). An air-drying method, in which somatic embryos were desiccated in an empty sealed Petri dish for 3–5d, gave rise to the best germination efficiency among the four desiccation methods tested: fast, slow, air, and KCl methods. The final percentage of moisture seems important since embyros over-dried by the fast and slow methods did not convert well into plants.     

A new semi-selective medium for the isolation of Xanthomonas axonopodis pv. dieffenbachiae, the etiological agent of anthurium bacterial blight

Aims:  Xanthomonas axonopodis pv. dieffenbachiae causes anthurium blight, which is regarded as the most threatening disease for the anthurium industry worldwide. The bacterium is listed as a quarantine pathogen in several regions, including Europe. We evaluated the use of Neomycin-Cephalexin-Trimethoprime-pivMecillinam 4 (NCTM4) medium for its isolation.
Methods and Results:  A total of 104 bacterial strains were inoculated onto NCTM4 and on the previously published Cellobiose-Starch (CS) and Esculin-Trehalose (ET) media. The strain collection included: the anthurium blight pathogen, Xanthomonas strains, for which false positive results are known to occur using serological identification-tests; other bacterial pathogens of anthurium; and representatives of bacteria that are commonly present in the anthurium phyllosphere. Media were evaluated following the ISO 16140 protocol for the validation of alternative methods.
Conclusion:  Growth of the anthurium blight pathogen was better on NCTM4 and ET media than on CS. NCTM4 provided a better repeatability. It also displayed a lower rate of false positive and false negative results when the pathogen was isolated from plant extracts.
Significance and Impact of the Study:  This study will lead to improved isolation protocols of the anthurium blight in official procedures. NCTM4 medium could also favourably be used in studies, which aim to further understanding of the biology and epidemiology of this pathogen.     

In vitro evaluation of a morphine polymeric complex: Flowability behavior and dissolution study

The purpose of this research was to perform a granulometrical and flow properties study of a morphine polymeric complex and determine the influence of 3 variables—particle size of complex, pH value, and ionic strength of the dissolution medium—on the dissolution behavior. The morphine-Eudragit L complex was produced in aqueous medium from morphine hydrochloride saturated solution and Eudragit L 30D diluted until 12% wt/vol and partially neutralized (40%). To determine the rheological behavior of the complex, several rheological tests were developed: bulk and tapped densities, Hausner ratio, angle of repose, and flow rate. The results corresponding to the technological study suggest that the 100- to 250-μm fraction can be considered as free flowing powder. In relation to the dissolution behavior of the complex, the results indicate that the ionic strength has been detected as the most influencing factor when values below physiological conditions are used. In conclusion, no technological problems for the production of further solid dosage forms are expected. Furthermore, no changes in the dissolution profiles of the complex have been detected when ionic strength values are inside the physiological range.     

小鼠肠道类器官三种不同条件培养基的比较研究

摘要 目的：比较三种不同条件培养基对小鼠类器官形态和增殖速度的影响。方法：取C57BL/6小鼠的小肠和结肠,EDTA法分离隐窝,以基质胶包埋,加入不同小鼠肠道类器官培养基培养7 天,使用光学显微镜记录和比较类器官形成率和出芽情况。随后进行二代类器官培养,使用TrypLE将类器官消化为单细胞,重新包埋和培养,使用光学显微镜记录和比较不同类器官培养基对二代类器官的培养效率。采用荧光定量PCR比较不同条件培养类器官中干细胞标志物Lgr5和分化标志物MUC2的表达。使用免疫荧光法检测类器官中ki-67的表达。结果：对于小肠类器官的培养,使用条件培养基1、IntestiCult条件培养基和L-WRN培养基培养结肠类器官的形成率分别为（18.2±4.5） %、（63.8±4.0） %和（82.1±8.4） %。其中使用IntestiCult条件培养基培养类器官的出芽率更高。对于结肠类器官的培养,使用条件培养基1、IntestiCult条件培养基和L-WRN培养基培养结肠类器官的形成率分别为（17.3±7.3） %、（58.0±6.1） %和（46.3±7.4） %。对于二代类器官的培养,IntestiCult条件培养基和L-WRN培养基都能够支持消化为单细胞后的二代类器官培养。干细胞标志物Lgr5和分化细胞（杯状细胞）标志物MUC2的表达无明显差异。使用L-WRN培养基的类器官ki-67阳性比例更高,增殖速度更快。结论：本研究比较了三种不同条件培养基对小鼠类器官形态和增殖速度的影响。经过对比,L-WRN培养基更有利于小鼠肠道类器官的形成和增殖速度。     

Effect of Bcl-2 Expression on Hybridoma Cell Growth in Serum-Supplemented, Protein-Free and Diluted Media

Two transfected hybridoma cell lines TB/C3-bcl2 (overexpressing the Bcl-2 protein) and TB/C3-pEF (control cell line), were compared in batch suspension cultures using a medium supplemented either with horse serum or with a protein-free, iron-rich supplement. The membrane intact index (percentage of cells with intact membranes determined by trypan blue staining) of the TB/C3-bcl2 cell line decreased much slower than that of the control cell line during the dying phase of the cultures. No significant difference in antibody, lactate and ammonia production as well as glucose and glutamine consumption was noted in the exponential phase of the experiments. Both cell lines were also compared in batch experiments using media diluted with saline to further investigate the effect of Bcl-2 under sub-optimal conditions. The Bcl-2 overexpressing cell line again exhibited a higher membrane intact index at increasing dilution steps. This revised version was published online in June 2006 with corrections to the Cover Date.     

Electron transfer reactions between quinols and quinones in aqueous and aprotic media

The pathways of redox equilibration of quinols and quinones have been investigated. The rate-limiting reaction involves the couple QH^?/QH^· of the reducing quinol and the couple Q^?/Q of the oxidising quinone. Three general mechanistic points may be surmised: (i) protonation/deprotonation reactions are not rate-limiting; (ii) all transfers occur in one-equivalent steps; (iii) electron transfers, but not hydrogen atom transfers, are the dominant features. In aprotic media, no rapid route of equilibration is available since the ionic species which are necessary for thermodynamically feasible routes of electron transfer cannot exist to any great extent. The relation of these results to models of biological quinone systems is discussed.     

Mutation and nuclear stage in Schizosaccharomyces pombe I. An experimental approach to the role of recombination in mutation induction

A reverse mutation system using G1 and G2 cells of Schizosaccharomyces pombe is described. In order to enable the system to deal with the problem of mutation dependence on recombination, tests were performed on (i) the homogeneity of cell populations with respect to nuclear stage; (ii) the fate of cells during post-irradiation incubation; (iii) the colony-forming ability of G1 and G2 revertants, and (iv) cell viability on the mutation plates. On the basis of the results, it is thought that, using this system, information can be obtained on the role of recombinational events in the process of mutation induction.     

The excision of pyrimidine dimers from the DNA of mutant and wild-type strains of Ustilago.

UV-induced pyrimidine dimers were excised from the DNA of wild-type and four mutant strains of Ustilago maydis. Excision was partially dose dependent. The kinetics of excision differed in recombination deficient strains (rec 1 and rec 2) from those found in a recombination proficient radiation-sensitive strain (uvs 3). At fluences above 100 J-m-2 excision was saturated in uvs 3 but not in rec 1 or rec 2. Fluences above 300 J-m-2 started to saturate excision in wild-type. pol1-1, a temperature-sensitive DNA polymerase mutant, was excision proficient at both the permissive (22 degree) and restrictive (32 degree) temperatures. Wild-type cells were observed to excise CC before CT or TT in high dose experiments.     

A novel combination of selective agents for isolation of Leptospira species

A novel combination of antimicrobial agents (sulfamethoxazole, 40 μg/mL; trimethoprim, 20 μg/mL; amphotericin B, 5 μg/mL; fosfomycin, 400 μg/mL; and 5-fluorouracil, 100 μg/mL) was developed for selective isolation of leptospires from contaminated samples. The growth of 16 microorganisms considered as possible contaminants during isolation of Leptospira were inhibited by this antimicrobial cocktail. In contrast, the growth of a smaller inoculum (10(1) cells per mL) of 25 Leptospira strains (representing 18 serovars/serogroups of 5 species) was not suppressed by this antimicrobial combination. This cocktail, after being incorporated into Leptospira growth medium (Korthof's), successfully detected leptospires in environmental soil and water. Based on the results, this selective medium has the potential to meet the existing need for an effective selective medium for the isolation of Leptospira.