In vitro cultivation and behavior of aortic endothelium cells in a low serum culture medium

Endothelial cells isolated from calf aorta were used in the subculture No. 4 to 10 for experiments to establish standardized and well reproducible conditions of cultivation. The cells can be cultivated in the commercial medium Eagle-MEM with following supplements: 0.1 g L-glutamine, 5.0 g peptone, 0.5 g serum albumin, and 10 ml (= 1%) bovine serum per 1000 ml medium (MEMPAS). With the aid of immunofluorescence technique the cell type specific marker Factor VIII antigen was shown to be localized especially in the perinuclear region of the cells. The cells were characterized with regard to their growth behaviour. Both, the MEMPAS and the Eagle-MEM with 10 per cent serum increases the cell number in the first 4 days of the exponential growth to the same values. The use of MEMPAS in connection with a strict cultivation regime from the deep frozen cell suspension to culture in scintillation vials guaranteed well reproducible conditions of cultivation. In 14 non-selected experiments distributed over a longer period of time it was found that with regard to the values of the cell number on the respective day the cultures can be divided into two groups, which differ with statistical significance. In further experiments it was possible to confirm this result. Medium, conditioned by endothelial cells (K-MEMPAS) increases the cell number and the growth rate. From these results it was concluded that endothelial cells of vessels are able to produce growth factors with self-stimulating effects. At this time the endothelial cell line is stored in deep frozen state up to the 25th subculture. The endothelial cells cultivated in the described standardized conditions are useful for screening of cell type specific factors with angiogenic activity.     

Automation of chromogenic substrate Limulus amebocyte lysate assay method for endotoxin by robotic system.

The chromogenic substrate Limulus amebocyte lysate (LAL) assay method for the detection of endotoxin was automated by a Zymate robotic system. The software developed enables the robot to automatically dilute a stock reference endotoxin standard (20,000 endotoxin units per ml) for the construction of a five-point standard curve, make sample dilutions to the proper testing concentration, and perform chromogenic substrate LAL assays in duplicate. The linearity of the standard curve and the endotoxin concentration in each sample are calculated and results are printed automatically. In 48 min the automated system assays three samples and a reference standard in duplicate along with a water blank. Sensitivity of the assay is a function of incubation time. The assay is linear (r greater than 0.99) in the region of 0 to 1.0 endotoxin units per ml or 0 to 0.2 endotoxin units per ml with incubation times of 10 or 16 min, respectively. The method can be made very sensitive, detecting as low as 0.003 endotoxin units per ml with 30 min of incubation. The precision of the assay method, determined by assaying an endotoxin reference solution eight times, is ca. 6%. The LAL reagent designed for gel-clot assay was modified for the chromogenic substrate assay. We describe the optimum conditions for the performance of the chromogenic substrate LAL assay and stability of the LAL reagent.     

Axenic Mass Cultivation of Entamoeba invadens and Cell Membrane Isolation

SYNOPSIS. A method for axenic mass cultivation of Entamoeba invadens is described. A known monophasic culture medium for E. histolytica was modified by mixing it with a medium for Acanthamoeba castellanii and adding ferric chloride to provide more particulate material. This results in a high number of cells per ml after a relatively short culture period.
A cell membrane fraction of such a mass culture of E. invadens can be prepared by simple disruption of the cells and by low speed centrifugation. Owing to the lack of membrane-bounded organellae in the amebic cytoplasm there are only 2 kinds of membranes to be separated: the plasmalemma and the vacuole membranes.     

Determination of L-ascorbic acid levels in culture medium: Concentrations in commercial media and maintenance of levels under conditions of organ culture

Summary The method of Deutsch and Weeks was modified to provide a reliable and reasonably quick method for assaying the L-ascorbic acid content of culture medium. The modified method was used to determine the decay of L-ascorbic acid under various conditions of culture and the concentration of the vitamin in commercially prepared media. The half-life of L-ascorbic acid in a modified New circulator gassed with 95% O₂+5% CO₂ was 1.5 hr.; and when gassed with 20% O₂+5% CO₂+75% N₂, about 2 hr. In Petri dishes gassed with 20% O₂+5% CO₂+75% N₂, the half-life of L-ascorbic acid was 0.9 hr. About 4% of the L-ascorbic acid was lost per day when medium was stored at 0°C and about 9% per day when stored at 5°C. When medium with an initial content of 300 μg per ml was stored at room temperature, the half-life was found to be 15.5 hr. The L-ascorbic acid in five commercially available media, which contain the vitamin in their formulations, was assayed immediately after their delivery to the laboratory. The values of L-ascorbic acid measured in these media were in all cases far lower than prescribed. A continuous-flow organ culture system has been designed which allows the provision of a relatively constant level of L-ascorbic acid to an explant by taking advantage of the slow oxidation of L-ascorbic acid at 0°C.     

Improved high-performance liquid chromatographic method for the determination of polycyclic aromatic hydrocarbon metabolites in human urine

A reversed-phase HPLC method with fluorescence detection was evaluated for utility in determination of urinary metabolites of polycyclic aromatic hydrocarbons as biomarkers of environmental exposure. The method, which was developed for use in studies of high-level occupational exposure, was found to be unreliable for relatively low-level environmental exposures. The method was modified to include quantitation by standard addition in order to compensate for matrix effects at levels as low as 0.1 ng/ml. The standard addition modification increased both qualitative and quantitative performance, with recovery of 1-hydroxypyrene spikes improved from 164% to 114% at 0.36 ng/ml. The modified method was successfully applied in an environmental exposure study.     

Retion of human apolipoprotein A-II by a mutant of Saccharomyces cerevisiæ

Summary A modified cDNA of mature human apolipoprotein A-II (apoA-II) was expressed by a sterol-uptake yeast strain (erg 10) of Saccharomyces cerevisiæ. ApoA-II cDNA was fused with a modified yeast alpha factor leader peptide coding sequence under phosphoglycerate kinase promoter control in a 2 micron-based plasmid construction. The use of cholesterol allowed apoA-II secretion in the culture medium. The mono and dimeric forms of apoA-II (approximately 3 micrograms per ml) were detected.     

A microbiological test system for determining dioxidine levels in biological fluids

A microbiological procedure for determining dioxidine concentrations in biological fluids with using E. coli AB 2472 rec A 16, a reparation deficient strain as a test organism is described. Cell suspension of the strain 24-hour culture is added to 1.2 per cent agar with Hottinger digest (140 mg per cent of amine nitrogen), 3 g/l of disubstituted sodium phosphate and 0.4 per cent of glucose cooled to 50 degrees C. 10 ml of the medium are added to every Petri dish with metallic cylinders put on the agar. After the medium solidification the cylinders are removed and 0.1 ml of the solution being tested is added to every well. The dishes are incubated for 24 hours under anaerobic conditions. The test system sensitivity is 0.2 microgram/ml of dioxidine. The relationship between the growth inhibition zone and the drug concentration is linear within dioxidine concentrations of 0.2 to 3.2 micrograms/ml.     

Production of Mevalonic Acid by Fermentation

The purpose of this investigation was to select microorganisms that produce substantial quantities of mevalonic acid and to develop an economic fermentation process. To screen for mevalonic acid-producing microorganisms, it was necessary to improve the method for the quantitative determination of this acid. The biological assay was modified by shortening the incubation time and simplifying the procedure. The principle of the assay is based on the essential mevalonic acid requirement of the organism Lactobacillus heterohiochii for growth. Screening was carried out by selecting high mevalonic acid-producing organisms from various type cultures. Endomycopsis fibuliger was chosen for medium development studies, and 939 mug of mevalonic acid per ml was produced in the culture filtrate after modifications of medium and fermentation conditions.     

ELISA法检测乙型脑炎减毒活疫苗中卡那霉素和庆大霉素的残留量

用ELISA试剂盒检测乙脑减毒活疫苗中卡那霉素和庆大霉素的残留量。以间接竞争ELISA法检测线性范围内加入高、中、低3种浓度的卡那霉素和庆大霉素,测定其在疫苗稳定剂、疫苗稳定剂10倍稀释溶液、试剂盒稀释液中的回收率,以及在10倍稀释乙脑减毒活疫苗中的回收率。高、中、低浓度的卡那霉素在疫苗稳定剂、疫苗稳定剂10倍稀释溶液、试剂盒稀释液中的回收率为101.40%～124.80%之间;原倍疫苗稳定剂对庆大霉素的测定有明显干扰,在原倍疫苗稳定剂中低浓度和中浓度的庆大霉素的回收率高达2280%和575%,但其它测定条件下回收率在90%～125%之间。在10倍稀释的乙脑疫苗中加入一定量的卡那霉素和庆大霉素,回收率分别为124%和103.25%。用10倍稀释法测定1人份规格的乙脑减毒活疫苗17批、5人份规格的乙脑减毒活疫苗19批。乙脑减毒活疫苗10倍稀释后,可用ELISA试剂盒检测卡那霉素和庆大霉素残留量。     

The effects of human leukemia inhibitory factor (hLIF) and culture medium on in vitro differentiation of cultured porcine inner cell mass (pICM)

Summary Isolation and maintenance of porcine embryonic stem (pES) cells have been hindered by the inability to inhibit differentiation of the porcine inner cell mass (pICM) in vitro. Culture conditions currently in use have been developed from mouse ES cell culture and are not effective for maintaining the pICM. Optimizing culture conditions for the pICM is essential. We have developed a grading system to detect changes in the differentiation status of in vitro cultured pICM. Porcine ICMs (Day 7) were isolated by immunosurgery and cultured for 4 d in either Dulbecco’s modified Eagle’s medium (DMEM)-based medium (D medium) or DMEM/Ham’s F-10 (1:1)-based medium (D/H medium) with or without human Leukemia Inhibitory Factor (hLIF, 1000 iu/ml). Colonies were photographed daily for morphological analysis. pICMs were categorized into one of two types based on their morphological profile: type A, nonepithelial or type B, epithelial-like. Eight investigators evaluated pICM differentiation using standardized differentiation profiles. Each pICM series was graded on a scale of 1 (fully undifferentiated) to 5 (fully differentiated) for each time point. Differentiation was verified by alkaline phosphatase activity, cytokeratin staining, and scanning electron microscopy. Neither hLIF nor culture medium delayed differentiation of pICMs (P=0.08 and P=0.25, respectively). The grading system employed was an effective tool for detecting treatment effects on differentiation of the developing pICM. These results demonstrate that hLIF cannot significantly inhibit differentiation of the pICM, and is unlikely to assist in porcine ES cell isolation. Future experiments utilizing homologous cytokines may prove more beneficial.     

Preparation and characteristics of protoplasts of Streptomyces kanamyceticus

To prepare actively regenerating protoplasts of S. kanamyceticus, the influence of the conditions of the mycelium cultivation, the culture age, lytic conditions, composition of the regeneration medium, the procedure of the culture inoculation to the regeneration medium and other parameters were studied. The study resulted in development of optimal conditions for preparation of S. kanamyceticus protoplasts in a number of 1.10(9) protoplasts per ml. The cultivation on the ST medium with 10 to 15% sucrose and addition of glycine up to 1% for 30 hours (the stationary growth phase) followed by treatment of the culture with lysozyme in an amount of 2 mg/ml for 1 hour at 32 degrees C provided preparation of up to 100% of actively regenerating protoplasts free of mycelium fragments. The size of the protoplasts increased up to 1.5 micron against the usually observed size of 0.7 to 1.0 micron with using modified lyzing buffer with 20% of sucrose according to the method recommended for S. erythreus. However, 50 to 70% of the protoplasts had point of linear regions in the cell walls, which suggested that spheroplasts were mainly forming and the phenomenon was associated with the characteristic properties of the strain cell wall structure.     

Lower sodium lactate in Whitten's medium improves in vitro developmental capacity of one-cell mouse embryos

A modification of Whitten's medium, involving a reduced content of Na-lactate syrup (0.2 ml/100 ml; 11.65 mM) and osmolarity (251 mOsm), was compared with normal Whitten's medium (0.37 ml/100 ml; 21.6 mM) for ability to support mouse embryonic development in vitro from one-cell to the blastocyst stage. In a pilot study utilizing 10 ICR donor female mice, in vitro developmental capacity (IVDC; percentage of fertilized one-cell embryos developing to blastocysts in vitro per female donor) was significantly enhanced by the modified medium (68.0 versus 24.0%; P<0.001). In the main study, utilizing 134 ICR and 17 ICR x C57BL/6J F(1) donor females, the modified medium supported increased IVDC for both ICR (67.9 versus 51.1%; P<0.001) and F(1) females (98.5 versus 89.4%; P<0.05). A large degree of among donor-female variation in IVDC was observed for both media in the ICR stock (SD = 30.0). The beneficial role of the reduction of Na-lactate in Whitten's medium may be related to an improved provision of energy requirements for first cleavage and/or a more suitable osmolarity for development.     

Variation in colony counts of total viable anaerobic rumen bacteria as influenced by media and cultural methods.

Volume and type of medium, carbohydrate concentration, carbohydrate ratios, and inoculum level were investigated as possible factors influencing total colony counts of anaerobic rumen bacteria obtained in roll tubes (18 by 150 mm). Colony counts were lower when the rumen fluid was clarified by centrifugation before inclusion in the medium; however, decreasing the volume of 40% rumen fluid glucose-cellobiose-starch-agar medium (RGCSA medium with 0.025% each of glucose and cellobiose and 0.05% starch, 4 ml per tube) was compared to the clarified rumen fluid medium and non-rumen fluid medium (medium 10) of Caldwell and Bryant (1966), 9 ml of each per tube. Total counts of rumen contents from sheep consuming four different types of rations were higher with the 4 ml of RGCSA medium than with the other two media. Dilution of the basal medium as a result of inoculum volume, as much as 1.5 ml per 4 ml of medium, did not appear to affect total counts. Colony counts and the simplicity of medium preparation and inoculation would favor the present method for routine use in estimating numbers of total viable anaerobic rumen bacteria, especially when large numbers of samples are involved.     

Determination of the potent antiprotozoal compound atovaquone in plasma using liquid-liquid extraction followed by reversed-phase high-performance liquid chromatography with ultraviolet detection

A specific and robust method is presented for the determination of atovaquone in plasma. Atovaquone is a potent antiprotozoal compound for use in immunocompromised patients who are intolerant of conventional therapies. The method involves a liquid-liquid extraction of the compound into hexane modified with 2% (v/v) isoamyl alcohol. The processed extracts are analysed by reversed-phase high-performance liquid chromatography with ultraviolet detection at 254 nm. The assay has a limit of quantification of 0.1 μg/ml and is linear between 0.1 and 50 μg/ml. The method has been applied to many clinical studies and has been demonstrated to be precise and accurate with high sample throughput. Atovaquone is not significantly metabolised in humans.     

Production of beta-fructofuranosidase with transfructosylating activity for fructooligosaccharides synthesis by Aspergillus japonicus NTU-1249.

Microbial beta-fructofuranosidases with transfructosylating activity can catalyze the transfructosylation of sucrose and synthesize fructooligosaccharides. Aspergillus japonicus NTU-1249 isolated from natural habitat was found to produce a significant amount of beta-fructofuranosidase with high transfructosylating activity and to have the potential for industrial production of fructooligosaccharides. In order to improve it's enzyme productivity, the medium composition and the cultivation conditions for A. japonicus NTU-1249 were studied. A. japonicus NTU-1249 can produce 83.5 units of transfructosylating activity per ml broth when cultivated in a shaking flask at 28 degrees C for 72 hours with a modified medium containing 80 g/l sucrose, 15 g/l soybean flour, 5 g/l yeast extract and 5 g/l NaCl at an initial pH of 6.0. The enzyme productivity was also optimized by submerged cultivation in a 5-litre jar fermentor with aeration at 1.5 vvm and agitation at 500 rpm. Under these operating conditions, the productivity of transfructosylating activity increased to 185.6 U/ml. Furthermore, the transfructosylating activity was improved to 256.1 U/ml in 1,000-litre pilot-scale fermentor. Enzymatic synthesis of fructooligosaccharides by beta-fructofuranosidase from A. japonicus NTU-1249 was performed in batch type by adding 5.6 units of transfructosylating activity per gram of sucrose to a 50% (w/v) sucrose solution at pH 5.0 and 50 degrees C. The yield of fructooligosaccharides was about 60% after reaction for 24 hours, and the syrup produced contained 29.8% (w/v) fructooligosaccharides, 15.2% (w/v) glucose and 5.0% (w/v) sucrose.     

Detection and production of verotoxin 1 of Escherichia coli O157:H7 in food.

Verotoxin 1 (VT1) is a recognized virulence factor of Escherichia coli O157:H7, a cause of severe food-borne disease. The public health significance of preformed verotoxin in food is unknown, and relatively little research has been done to determine the production of VT1 in food. The purposes of this study were to develop a sensitive method to detect VT1 in milk and in ground beef and to determine the conditions for VT1 production in these foods. A sandwich enzyme-linked immunosorbent assay in which we used VT1-specific monoclonal antibody 9C9F5 as the capture antibody and a rabbit polyclonal antibody raised against VT2 as the detection antibody was developed for the detection and quantification of VT1 in milk and in ground beef. The enzyme-linked immunosorbent assay was sensitive to a minimum of 0.5 ng of VT1 per ml of milk and 1.0 ng of VT1 per g of ground beef. The greatest amount of VT1 detected in milk (306 ng/ml) was detected in samples that were incubated at 37 degrees C with agitation (160 rpm) for 48 h. Very little toxin (1 ng/ml) was produced at 25 or 30 degrees C within 96 h. VT1 production was greater in ground beef than in milk; 452 ng of VT1 per g was produced in beef at 37 degrees C in 48 h. Relatively little VT1 was produced in beef within 96 h at 25 and 30 degrees C (2.1 and 9.8 ng of VT1 per g, respectively). Our results indicate that ground beef is a better medium for VT1 production than milk.(ABSTRACT TRUNCATED AT 250 WORDS)     

A simple and reliable method for monitoring platelet serotonin levels in rats

A simple and reliable method for individual monitoring of platelet serotonin in rats is developed. Platelet-rich plasma is prepared under standardized conditions from 1 mL of venous blood and the platelets are quantitatively separated by a highly reproducible procedure. Platelet serotonin content is determined spectrophotofluorometrically and the results are comparatively expressed per standardized platelet rich plasma sample (1.01 +/- 0.18 microgram), per mg of platelet protein (1.57 +/- 0.15 microgram) and per 10(9) platelets (2.16 +/- 0.38 micrograms). Normal distribution of platelet serotonin levels in a sample of 338 animals is shown. By use of the described method, the intraindividual stability of platelet serotonin concentration in rats is demonstrated for the first time.     

Detection and production of verotoxin 1 of Escherichia coli O157:H7 in food.

Verotoxin 1 (VT1) is a recognized virulence factor of Escherichia coli O157:H7, a cause of severe food-borne disease. The public health significance of preformed verotoxin in food is unknown, and relatively little research has been done to determine the production of VT1 in food. The purposes of this study were to develop a sensitive method to detect VT1 in milk and in ground beef and to determine the conditions for VT1 production in these foods. A sandwich enzyme-linked immunosorbent assay in which we used VT1-specific monoclonal antibody 9C9F5 as the capture antibody and a rabbit polyclonal antibody raised against VT2 as the detection antibody was developed for the detection and quantification of VT1 in milk and in ground beef. The enzyme-linked immunosorbent assay was sensitive to a minimum of 0.5 ng of VT1 per ml of milk and 1.0 ng of VT1 per g of ground beef. The greatest amount of VT1 detected in milk (306 ng/ml) was detected in samples that were incubated at 37 degrees C with agitation (160 rpm) for 48 h. Very little toxin (1 ng/ml) was produced at 25 or 30 degrees C within 96 h. VT1 production was greater in ground beef than in milk; 452 ng of VT1 per g was produced in beef at 37 degrees C in 48 h. Relatively little VT1 was produced in beef within 96 h at 25 and 30 degrees C (2.1 and 9.8 ng of VT1 per g, respectively). Our results indicate that ground beef is a better medium for VT1 production than milk.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gas Consumption and Growth Rate of Hydrogenomonas eutropha in Continuous Culture

The bacterium Hydrogenomonas eutropha is under consideration for use in a regenerative life-support system for manned space missions of long duration. A 4-liter continuous culture unit containing the organism was operated for a period of 272 days under autotrophic environmental conditions. The best steady-state run achieved with this unit was observed over a 22-day time interval after 181 days of operation. During this time, the culture consumed an average of 22.9 +/- 2.0 ml of carbon dioxide per min, 38.1 +/- 3.3 ml of oxygen per min, and 128.5 +/- 10.6 ml of hydrogen per min. It required 18.7 +/- 1.2 liters of fresh nutrient medium per 24 hr to maintain a constant, preestablished cell population of 1.65 g (dry weight) per liter. The ratio of consumption of carbon dioxide, oxygen, and hydrogen varied from 1:1.2:4.5 to 1:1.9:6.6, with an average of 1:1.7:5.7. Based on these values, approximately 60 liters of the culture would be necessary to balance the gas exchange of one man.