Effects of Temperature,Shoot Age,and Medium on Gall Induction by Subanguina picridis in Vitro

The influence of temperature, shoot age, and medium on gall induction by Subanguina picridis on Russian knapweed (Acroptilon repens) was examined in vitro. The optimal temperature for gall formation was 20 C. Gall induction was delayed as the temperature decreased, and decreased as shoot age increased. Bud primordia (0-day-old shoots and 5-day-old shoots) with an average length of 4.2 mm and 7.9 mm were the most suitable tissues for nematode development and gall formation. Gall formation was more effective on B5G medium than on MSG. Young shoots under slow growth were most suitable for mass rearing of S. picridis.     

Defined media optimization for growth of recombinant Escherichia coli X90

An optimized, defined minimal medium was developed to support balanced growth of Escherichia coli X90 harboring a recombinant plasmid. Foreign protein expression was repressed in these studies. A pulse injection technique was used to identify the growth responses to nutrients in a chemostat. Once the nutrients essential for growth had been identified, the yield coefficients for individual medium components. These yield coefficients were used to develop an optimized, glucose-limited defined minimal medium that supports balanced cell growth in chemostat culture. The biomass and substrate concentrations follow the Monod chemostat model. The maximum specific growth rate determined in a washout experiment is 0.87 h(-1) for this strain in the optimized medium. the glucose yield factor is 0.42 g DCW/g glucose and the maintenance coefficient is zero in the glucose-limited chemostat culture. (c) 1993 John Wiley & Sons, Inc.     

南极乔治王岛冰锥洞微生物培养探索

【目的】南极洲具备独特的环境和相对的生物地理隔离,南极洲各类生境中蕴藏了大量尚未培养和难培养的微生物,也是新颖微生物物种的重要来源之一。本研究以南极冰锥洞这类特殊生境为研究对象,通过培养条件的多样化提升南极微生物的培养率和多样性,揭示南极冰锥洞可培养微生物类群多样性,为该环境可培养微生物功能研究奠定基础,也为南极极端环境未培养微生物的培养方法提供借鉴。【方法】通过采用不同培养基添加复苏促进因子(resuscitation promoting factor, Rpf)的方式,提高南极柯林斯冰盖冰锥洞生境中微生物的可培养率,探究该生境中微生物的多样性。采用4种不同营养水平的培养基,平行添加Rpf进行菌株培养,经分离纯化与16S rRNA基因鉴定,分析冰锥洞可培养微生物的多样性及培养条件对多样性的影响。【结果】本研究共分离培养细菌407株,涵盖5个门、18个科、29个属,其中：放线菌门(Actinomycetota)为优势门,占72.73%;微杆菌科(Microbacteriaceae)为优势科,占69.78%;Lacisediminihabitans属为优势属,占45.70%。从培养基效果...     

Lysozyme as a regulator of interleukin-2-activated lymphocyte proliferation

Summary Lysozyme at 1 to 100μg/ml of exposure levels augmented or inhibited proliferative response of human peripheral blood lymphocytes stimulated with interleukin-2 (IL-2). This contradictory effect of lysozyme depended on IL-2 concentration, activating state of lymphocytes, addition time of lysozyme, and serum existence. Lymphocytes increased their IL-2-mediated proliferating ability in response to lysozyme when stimulated with less than suboptimal concentration of IL-2. Lymphocyte activation with anti-CD3 antibody changed the augmented proliferative response into the inhibited response by lysozyme addition whereas elimination of MHC class II molecule-expressing cells augmented the response. Addition of lysozyme within 1 h after IL-2 exposure was most effective in promoting the proliferation whereas additions after 16 to 24 h were ineffective or inhibitory. Addition after longer than 24 h inversely restored the proliferative response. Serum seemed to retard lysozyme action because either sequential serum addition 1 h after exposure of IL-2 and lysozyme to cells or exposure of IL-2 and serum after pretreatment of cells with lysozyme changed the proliferative responsiveness from inhibition into augmentation. Thus lysozyme may regulate lymphocyte proliferation responding to a magnitude of antigenic stimuli and to the progression of cellular events that periodically occur.     

Cultures of bovine tracheal epithelium with differentiated ultrastructure and ion transport

Summary Tracheal epithelial cells were grown on Nuclepore filters coated with human placental collagen. When grown immersed in medium containing fetal bovine serum, cells displayed an undifferentiated ultrastructure (no cilia and a cell height of ∼ 10 μm). Short-circuit current (I_sc) was approximately 1/10 that of the native epithelium. By contrast, when grown in hormonally defined, serum-free medium with an air interface, cells showed I_sc equal to or greater than the original tissue, possessed cilia, and had a cell height of ∼ 50 μm. Responses in I_sc to mediators were similar to those of the original tissue, but differed from those of dog or human tracheal epithelium. Given the ready availability and low cost of the native tissues, bovine tracheal cultures grown in serum-free medium with an air interface should prove useful in studies of airway epithelial physiology.     

Growth of cells in a new defined protein-free medium

The development of a new stable synthetic serum replacement (SSR) is described, which allows the cultivation of mammalian cells in a defined, protein-free medium containing only dialyzable components. With a low concentration of insulin (RPMI-SR2 medium), growth rates of the transformed cell lines L929, HELA S3, and the hybridoma 1E6 were comparable to growth rates obtained with a serum-containing medium. The same medium also supported long-term cultivation of non-dividing mouse macrophages. The main principle of SSR is a metal ion buffer containing a balanced mixture of iron and trace metals. Stability against precipitation of important metals is achieved by the combined use of EDTA and citric acid as chelating agents. Efficient iron supply is mediated through the inclusion of the compound Aurintricarboxylic acid as a synthetic replacement for transferrin. SSR also contains a growth-promoting surfactant, Pluronic F68. Thus SSR provides a general foundation for growth and differentiation normally provided by serum.Limitations of other serum-free medium designs are discussed here: 1) the inability of transferrin to chelate all metals in the medium; and 2) the use of inorganic iron salts or iron citrate as an iron supplement leads to rapid precipitation of iron hydroxide in the medium. Both these problems are solved in the design of SSR.     

Substitution of transferrin by FeCl3 in the development of a low foetal calf serum concentration medium for KB-26.5 hybridoma cell line

KB-26.5, a murine hybridoma cell line producing an IgG₃ monoclonal antibody used in blood type determination, primarily adapted to grow at 5% foetal calf serum (FCS) concentration has been adapted to grow at 0.5% FCS, maintaining its ability to produce antibodies at the same level. In the final step of adaptation, the addition of insulin, transferrin, ethanolamine and selenium to the media formulation was studied, using factorial assay techniques to check the effect of the different compounds and to optimize their required level for satisfactory growth and antibody secretion. KB-26.5 cells required only 20 g/ml of transferrin to adapt to 0.5% FCS medium. Furthermore, transferrin could be substituted by FeCl₃, at a relatively low level of 2 g/ml. Maximum cell density decreased by 31.5% in spinner flask test, but the antibody titer was maintained, thus the specific productivity increased. However, inoculum size had to be increased three-fold with 0.5% FCS medium in order to assure cell growth.     

基于响应面分析的高抗原活性猪水肿病大肠杆菌疫苗培养基的研制及效果评价

【背景】猪水肿病大肠杆菌引发的疾病造成了很大的危害,但现有培养基存在培养密度低的问题。【目的】研制出高抗原活性猪水肿病大肠杆菌疫苗培养基。【方法】以常用的市售猪水肿培养基为对照,通过单因素试验、爬坡试验(Plackett-Burman, PB)、响应面(Box-Behnken, BB)试验对猪水肿培养基进行响应面优化,得到猪水肿培养基最优配方。以响应面试验得到的培养基培养猪水肿病大肠杆菌,评价不同培养时间点菌株的抗原活性,制作灭活疫苗,进行动物免疫保护试验。【结果】对研制的培养基进行扩大培养验证,发现扩大培养得到的菌株活菌数可达5×10⁹ CFU/mL以上,约为对照组的2倍。制备的灭活疫苗效价可达1:140 000,并在9 h时抗原蛋白效价达到最高。【结论】本研究研制出的疫苗培养基显著提高了猪大肠杆菌菌体密度,并可提高菌体抗原活性,为猪水肿病灭活疫苗的制备提供了技术指引。     

Normal rat kidney proximal tubule cells in primary and multiple subcultures

Summary Anin vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys and separating the cortex, the cortical tissue is digested with the enzyme DNAse-collagenase (Type I) resulting in a high yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency in a serum-free, hormonally defined medium. The cell yield can be increased by transfering the conditioned medium on Day 1 to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces than on bovine collagen Type I coated surfaces. The culture medium was a 1∶1 mixture of Ham’s F-12 and Dulbecco’s modified Eagle’s medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal growth factor, and glutamine. The RPT cells became confluent in 7–10 d, at which point they could be subcultured by trypsinizing and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The cells were histochemically positive for alkaline phosphatase and γ-glutamyl transpeptidase activity and synthesized the intermediate filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence, the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture model will be a valuable tool for substrate uptake and nephrotoxicity studies.     

Continuous hybridoma culture in a low-protein serum-free medium supplemented with liposomes

A homemade serum-free medium containing a low protein level under 0.1 g l⁻¹ has been proved to support long-term cultures of VO 208 hybridoma cells successfully up to 50 days. The low protein level was achieved by supplying the lipids through liposomes containing cholesterol, oleic acid, - dipalmitoyl phosphatidylcholine, and bovine serum albumin. The influence of the liposome content in the feeding medium was studied in a continuous culture performed with step variations of the liposomes level, from 7.5 to 30 ml l⁻¹. The cell density decreased at the highest liposomes content while it became higher with 7.5 or 12 ml l⁻¹ of liposomes. For each step variation appeared a transitory activation of the specific rates of nutrient consumption, metabolite production and antibody secretion, as well as a transitory decrease of the specific cell growth rate. The overall structure of the antibodies was not affected during the culture.