Earle's Balanced Salt Solution Research Articles

Freezing Excysted Coccidial Sporozoites

Excysted sporozoites of Eimeria adenoeides, E. mivati, and E. tenella were placed in media containing different concentrations (2.5, 5, 7.5, 10, 12.5, and 15%) of dimethyl sulfoxide (DMSO). In the absence of freezing, survival of sporozoites was better at the lower concentrations. However, after sporozoites were frozen with a cooling rate of 1?/min from 25 to 26 C to -30 C and 10?/min from -30 C to -80 C, survival was better at the higher concentrations. With 10% DMSO for E. tenella and 12.5% for E. adenoeides and E. mivati, sporozoites were frozen using (1) equilibration periods of different lengths (20 to 50 min) and (2) different cooling rates (1, 3, and 5?/min) from the beginning of the procedure to the freezing point and from freezing to -30 C. The rate from -30 to -80 C was always 10?/min. Best results with all 3 species (more than 70% survival) were obtained when the equilibration period was 45 to 50 min and the cooling rate from the freezing point to -30 C was 1?/ min. The cooling rate from beginning to the freezing point was not important; no differences in survival were found after using different rates, and sporozoites withstood an abrupt temperature drop to 4 C. Attempts to increase the survival of sporozoites in media containing 2.5, 5.0, and 7.5% DMSO by increasing the equilibration period to as long as 2.5 hr were unsuccessful. Kouwenhoven (1967), Norton and Joyner (1968), Norton et al. (1968), Doran and Vetterling (1968a, 1969), and Doran (1969) have reported on the low temperature preservation of coccidia. Sporozoites within intact oocysts were not viable after freeze-thawing. However, sporozoites within sporocysts released from oocysts and excysted sporozoites were viable. This report pertains to the (1) concentration of freezing protectant, (2) equilibration period, and (3) cooling rates during freezing of excysted Eimeria adenoeides, E. mivati, and E. tenella sporozoites. MATERIALS AND METHODS Oocysts were collected from droppings of chicks or turkey poults during the first 72 hr of the patent period. They were recovered and sporulated by the method of Vetterling (1969). All oocysts were from suspensions that had been rendered bacteria-free for cell culture work by the method of Jackson (1964). They were stored in sterile Ringer's solution at 3 to 6 C and were less than 9 weeks old when used. Sporocysts were released from oocysts by grinding (Doran and Vetterling, 1968b) and suspended in trypsin-bile solution (Doran and Vetterling, 1967) at 43 C until 85 to 90% of the sporozoites within released sporocysts had excysted. Sporozoites were concentrated by centrifugation and placed in media to which either 2.5, 5, 7.5, 10, 12.5, or 15% dimethyl sulfoxide (DMSO) had been added. The medium for E. adenoeides and Received for publication 20 June 1969. E. tenella was Medium 199 with Hank's balanced salt solution (BSS) + 5% chicken serum; for E. mivati, 93% Earle's BSS, 5% lactalbumin hydrolysate (2.5% solution in Earle's) and 2% calf serum. All sporozoite suspensions were adjusted to pH 7.0 to 7.2 with 0.5 N NaOH or HC1. Those for freezing contained 500,000 sporozoites/ml. They were dispensed in 3-ml amounts into 10-ml roundbottom, screw-top tubes. Counts were made directly from one of the tubes in each group immediately before they were placed in the freezer. Suspensions to be used for determining survival of unfrozen sporozoites contained 250,000/ml. They were in 10-ml amounts within 50-ml, screw-top tubes at 43 C. Counts were made immediately after sporozoites were suspended in media and at 0.5-hr intervals thereafter. All counts were made at room temperature with a hemocytometer at a magnification of 156 X. Only those sporozoites that were refractive were counted as living. These were found not to stain with 1% nigrosin and to show motility when examined at 37 to 43 C. Freezing was carried out in a biological freezer equipped with controls for altering the rate of cooling and an override system for eliminating latent heat of fusion. A strip chart recorder monitored the rate of cooling. Unless otherwise noted, cooling was started at room temperature (25 to 26 C) and the rate of cooling from beginning to -30 C was 1?/min. The cooling rate was always 10?/min from -30 to -80 C. There was never more than 1? latent heat. Immediately after reaching -80 C, tubes containing sporozoites were removed and placed in liquid nitrogen vapor (Doran and Vetterling, 1968a). They were either thawed and examined on the same day or .stored for 1 to 4 days before examination. Differences in survival rate during these short intervals were not anticipated; Doran

Hepatic l-glutamate dehydrogenase: Changes in activity and kinetic properties in response to small molecules

A marked increase in the activity of l-glutamate dehydrogenase was observed when liver slices were incubated in the presence of malate, lactate, glutamate, or α-ketoglutarate. This increased activity was insensitive to both actinomycin D and puromycin. The GDH of perfused rat liver and of liver homogenates was also activated when Earle's balanced salt solution containing malate was utilized as perfusate or diluent. The endogenous and activated forms of the enzyme differed in several kinetic properties (Km, analogue reactivity, and citrate effects), but not their electrophoretic properties. The increased GDH activity and concomitant complete loss of AlaDH activity suggest that malate is effecting a conformational change in the enzyme subunits.

Development of first-generation schizonts of Eimeria bovis in cultured bovine cells.

SYNOPSIS. Monolayer primary and secondary cultures of embryonic bovine kidney, spleen, intestinal and testicle cells, and secondary cultures of embryonic bovine thymus, maintained in lactalbumin hydrolysate, Earle's balanced salt solution and ovine serum were observed for a maximum of 21 days after inoculation of E. bovis sporozoites. The sporozoites entered the cells in all of these cultures but underwent development only in primary cultures of kidney and intestinal cells and in secondary cultures of kidney, spleen, thymus, intestinal, and testicle cells. In acellular media, the sporozoites retained motility no longer than 21 hr. In the cell cultures, free motile sporozoites were seen for as long as 18 days after inoculation. Sporozoites entered cells anterior end first; the process of penetration required a few seconds to about a minute. Sporozoites were also observed leaving host cells. Intracellular sporozoites were first seen 3 min after inoculation; they were observed at various intervals up to 18 days after inoculation. In transformation of sporozoites into trophozoites a marked change in size and appearance of the nucleus took place before the change in shape of the body occurred. Trophozoites were first found 7 days after inoculation, multinucleate schizonts after 8 days, and schizonts with merozoites after 14 days. Schizonts containing merozoites were seen only in kidney, spleen, and thymus cells. The mature schizonts were smaller and represented a much lower proportion of the total number than in comparable stages of infections in calves. Schizonts with many nuclei occurred in intestinal cells; the most advanced stage seen in testicle cells was the binucleate schizont. Nuclear and cytoplasmic changes were observed in the infected cells.

Routine Establishment of Serial Lines of Hamster Embryo Cells Transformed by Adenovirus Type 12<xref ref-type="fn" rid="FN2">2</xref>

Whole hamster-embryo cells consistently underwent morphological alterations after 14–23 days in vitro when inoculated in suspension with adenovirus type 12, strain Huie, at multiplicities of 7 or more and incubated in a humidified 5% CO2 atmosphere at 37 C. Morphological changes were never observed when cells were incubated in the absence of 5% CO2. Altered cells were successfully cultivated for long periods and 12 serial lines were established in 12 attempts when medium NCTC 109 supplemented with 10% agamma calf serum was used. Attempts to establish serial lines on minimum essential medium in Earle's balanced salt solution with added serum were unsuccessful. Serial lines of hamster embryo cells transformed by adenovirus type 12 (ad-12) had the following inheritable characteristics: 1) small polygonal cells of uniform morphology; 2) accelerated growth rate; 3) loss of contact inhibition; 4) presence of ad-12 “T” antigen; and 5) transplantability into newborn and weanling hamsters. Virus could not be recovered from any cell line by conventional techniques. Of the 6 oncogenic ad-12 strains tested, only the prototype strain Huie was able to induce permanent morphological changes in hamster cells.

The effect of calcium on cell lines derived from adenovirus type 12-induced hamster tumors

Two cultures derived from adenovirus type 12-induced tumors have been shown to form clumps and ball-like aggregates upon continuous passage in media containing Earle's balanced salt solution. This effect is caused by calcium and can be reversed by removing calcium from the medium.

Leukocytes cultured from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypotonic KCl.

Leukocytes were cultured from 0.2 ml of whole blood inoculated into 5 ml portions of a medium consisting of Eagle's basal amino acids and vitamins at double strength in Earle's balanced salt solution brought to pH 7.0 with 7.5% NaHCO3, and containing additives: glutamine, 2 mM; penicillin, 100 units/ml; streptomycin, 100 μg/ml; phenol red, 7 μg/ml; fetal or newborn agammaglobulin bovine serum, 15%; phytohemagglutinin M, 2%; and U.S.P. heparin sodium, 20,000 units/liter. Cultures were incubated in closed 60 × 28 mm screw-cap vials, in a gas phase initially of room air, for 3 days at 37 C, with colchicine to make 0.2 μg/ml added for the final 3-5 hr. After incubation, the cells were separated from the medium by centrifugation, the medium replaced by 0.075 M KCI plus 16 U.S.P. units/ml of heparin sodium at 37 C, cells resuspended and allowed to incubate 10 min. Removal of the hypotonic KCI was followed by fixation in methanol-acetic acid, 3:1 (changed twice), spreading cells on slides by the air-drying metho...

THYROTROPIN STIMULATION OF THYROXINE SYNTHESIS IN ISOLATED THYROID CELLS TREATED WITH PUROMYCIN.

The in vitro incorporation of I into thyroxine and its precursors, monoiodoand diiodotyrosine, was observed in isolated bovine thyroid cells. This utilization of I was stimulated 2to 3-fold when the cells were incubated in the presence of 0.1 U of TSH/ml. Perchlorate ion added at a concentration of 2 mM completely eliminated iodide-concentrating activity by the cells, but did not significantly impair the stimulatory action of TSH on the incorporation of I into thyroxine and the iodotyrosines. Enhanced iodide-concentrating activity appears not to be required for this action of TSH on thyroxine formation. (Endocrinology 75: 968, 1964) A PRONOUNCED stimulatory action of J\. TSH in vitro on the incorporation of I into T4 and into iodotyrosines by isolated thyroid cells has recently been described (1, 2). This enhanced utilization of I was observed without a concomitant increase in iodide-concentrating activity as reflected by intracellular to extracellular I-iodide concentration (C/M) ratios. In fact, C/M ratios and the rate of iodide accumulation were consistently depressed by added TSH (2). A similar dissociation of the effects of TSH on iodide concentration and iodoprotein formation has been demonstrated in hypophysectomized rats by Halmi et at. (3). Four hr after injection of TSH, thyroidal uptake (and presumably organic binding) of I was increased to more than twice that of control hypophysectomized rats. At the same time, thyroid to serum I-iodide concentration ratios (observed in hypophysectomized rats injected with propylthiouracil) were depressed from a mean control value of 5 to a mean value of 2.1 by the same TSH treatment. From the results of our in vitro experiments with thyroid cells, and Halmi's in vivo observations, it may be inferred that TSH has a Received May 25,1964. Aided by a Research Career Development Award and Research Grant AM-06439 from the USPHS. 1 Abbreviations: TSH for thyroid-stimulating hormone, T4 for thyroxine, MIT for 3-iodo-Ltyrosine and DIT for 3,5-diiodo-L-tyrosine. direct stimulatory action on thyroxine formation that is not dependent upon increased intracellular concentration of iodide. Further support for this conclusion is provided in the present studies in which TSH stimulation of thyroxine (and iodotyrosine) formation was observed in thyroid cells treated with perchlorate to eliminate iodide-concentrating activity. Materials and Methods Procedures for the isolation, incubation and chromatographic analysis of thyroid cells have been described previously (1, 2, 4). For each experiment 140 g of minced bovine thyroid tissue was trypsinized to yield approximately 3 ml of cells. The cells were suspended in 25 volumes of Earle's balanced salt solution, and 5 ml aliquots of this homogeneous cell suspension were distributed into 25 ml Erlenmeyer flasks. In these experiments, all the specimens were preincubated for 15 or 30 min in the presence of 2 mM perchlorate. Then carrier iodide (4X10~M), I-iodide (5 /xc/ml) and TSH (where appropriate) were added and the incubations were continued for 3 hr. After incubation, the cells were recovered by centrifugation for 5 min at 50 X g, homogenized in 1 or 2 ml of a reagent consisting of 0.5% (w/v) of Pronase (Calbiochem), 30 mM Tris (pH 7.5), 2 mM MnS04 and 2 mM 1-methyl, 2-mercaptoimidazole (Tapazole), and hydrolyzed at 37 C for 8 hr. 200 /J aliquots of the resulting hydrolysates were chromatographed on Whatman No. 3 MM paper with a collidine-3N NH40H (100:37) solvent system. The iodide-concentrating activity of each cell preparation was observed and expressed in terms of the ratio of intracellular to extracellular I-

STIMULATORY EFFECT OF THYROTROPIN ON SYNTHESIS OF THYROXINE BY ISOLATED THYROID CELLS.

Iodide transport and thyroxine synthesis were studied in isolated cells prepared by trypsinization of bovine thyroid glands. When the cells were incubated in media containing 0.2 unit of thyrotropin (TSH) per ml, incorporation of I into thyroxine (and iodotyrosines) was stimulated 2to 4-fold, while no enhancement of iodideconcentrating activity could be detected. (Endocrinology 74: 304, 1964) D cells, obtained from ovine thyroid tissue by trypsinization, have been shown to retain the capacity to concentrate iodide, and to incorporate I-labeled iodide into iodotyrosines and iodothyronines (1). In the present study, the effects of added thyrotropin on these functions of the isolated cell preparation were investigated. Materials and Methods Dispersed thyroid cells were prepared by means of the continuous-flow trypsinization technique described previously (1). The following modifications of the technique were used in the present study: (a) bovine thyroid tissue was used instead of ovine tissue, (b) Earle's balanced salt solution was used in place of Hank's solution to prepare the trypsinizing reagent, (c) the freshly collected cells were washed with a dilute DNase solution to eliminate sliminess so that homogeneous cell suspensions could be obtained. In each experiment 2-3 ml of a fresh thyroid cell preparation was suspended in 50 ml of Earle's solution containing 4XlO~ M Nal labeled with I. 5 ml aliquots of such cell suspensions, containing 0.2-0.3 ml of cells, were delivered into 25 ml Erlenmeyer flasks and incubated under 95 % O2-5 % CO2 with shaking (90 oscillations/min) at 37 C in a Dubnoff water bath. After incubation, the contents of the flasks were transferred to Shevky-Stafford type centrifuge tubes, and the cells were sedimented by centrifugation for 5 min at 50 Xg in an International centrifuge maintained at a temperature of 5 C. Packed cell volumes were read to the nearest 0.01 ml, and then the supernatant media were drawn off. Iodine-concentrating activity was expressed as C/M ratios, I-iodide per ml cells I-iodide per ml medium The formation of organically bound I was prevented by incubating in the presence of 0.002 M Tapazole, so that I assays performed on packed cell pellets and aliquots of incubating media provided a direct measure of I-iodide distribution. However, the C/M values presented below are not corrected for the volume of the media trapped in the packed cell pellets. Extracellular space determined with C-inulin and I-albumin amounted to 49-60% of the packed cell volumes; hence, the true intracellular I-iodide concentrations were probably about twice those indicated by the uncorrected C/M values. In the chromatographic studies, I-labeled cells were homogenized in 10 volumes of 0.03 M tris buffer solution (pH 7.5) which contained 0.5 % (w/v) of Pronase. After incubation for 6-9 hr at 37 C, 200 /J aliquots of the hydrolysates were chromatographed on Whatman No. 3 MM paper in collidine-3N NH40H (1). r e labeled components were located by radioautography and assayed by means of a well-type Nal crystal, scintillation detector. Received September 4, 1963. Aided by USPHS Grant AM-06439. 1 Worthington desoxyribonuclease I. 2 Purchased from Fisher Scientific Co. 3 Abbreviations: Tapazole for 1-methyl, 2mercaptoimidazole; TSH for thyroid-stimulating hormone; MIT for 3-iodotyrosine; DIT for 3,5-diiodotyrosine; T4 for thyroxine; and tris for tris (hydroxymethyl) amino methane. 4 Pronase, Streptomyces griseus protease, was obtained from California Corporation for Biochemical Research.

ARBOR群ウイルスの化学的症状に関する研究

In the first paper of this serial works, the author reported that crude Japanese B encephalitis virus preparations were successfully purified by techniques of column chromatography with ECTEOLA-Cellulose ion-exchanger or by fluorocarbon deproteinization.In attempt to develope these findings on Japanese B encephalitis, the present paper describes that some other arbor viruses and poliovirus were also partially purified by cellulose chromatography or fluorocarbon deproteinization.Results obtained are as follows:1) The viruses used were dengue fever type 1, Mochizuki strain; yellow fever, strain 17 D; and Western equine encephalitis, Rockefeller Institute stock strain. The viruses were also grown in trypsinized hamster-kidney cell cultures. The concentration of each crude virus in infected fluid was about 105.0 (mouse intracerebral LD50 per 0.02ml) in the case of dengue fever, 104.0 in yellow fever, and 107.0 in Western equine encephalitis respectively.Poliovirus, strain MEF-1, was used for comparison and grown in HeLa cell culture too. HeLa cells were grown in culture bottles with medium consisting of 2% yeast extract, 0.5% lactalbumin hydrolysate in Earle's balanced salt solution (YLA medium), and 20% bovine serum.2) Culture fluid harvested from the infected cultures, showing characteristic cellular degenerization, was centrifuged at 3, 000r.p.m./min. for 10 minutes to remove crude debris masses. The supernatant fluid was loaded onto a column containing ECTEOLA-Cellulose and eluted with 0.07M phosphate buffer solution (pH 7.2). Some supernatant fluid was treated with fluorocarbon. In the case of yellow fever and Western equine encephalitis viruses, 100% of the original viruses was practically recovered and 90-93% of the nitrogen contained in the original virus materials was removed. Nitrogen contents of purified virus suspensions, as measured by the micro-Kjeldahl method, ranged mainly from 0.1 to 0.2mg/ml.