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BHK21 cells cultured in minimal essential medium (Eagle) supplemented with 10% dialyzed fetal calf serum did not grow as they did in whole serum containing medium. Logarithmic growth was, however, initiated after a lag period, the length of which was dependent upon the cell density: medium volume ratio. The quiescent cells conditioned the medium during this lag period, and growth stimulation was apparently due to the release of serine into the medium. Cells cultured in 10% dialyzed serum plus the low molecular weight fraction of serum (serum dialysate), grew with kinetics similar to cells cultured in serum containing medium. When serum dialysate was chromatographed on Bio-gel P-2 the growth promoting activity eluted with the amino acids. Each of the non-essential amino acids was tested for its ability to stimulate the growth of cells in 10% dialyzed serum. Serine was capable of stimulating cell growth to the same extent as 10% serum dialysate and its concentration optimum was similar to its concentration in 10% serum dialysate. The remaining non-essential amino acids were either slightly stimulatory or had no effect on cell growth. Shifting a logarithmically growing population of cells to serine-free medium resulted in the accumulation of 95% of the cells in the G1 phase of the cell cycle within 24 h. Escape from the G1 block could occur if serine was added to the medium or if the cells were allowed to condition the medium. Entry of cells into S phase after the addition of 0.05 μmoles/ml of serine followed a 4–6 h lag and 80% of the cells were synthesizing DNA 12 h after shift-up.

3T3 cells do not grow in Methocel suspension culture, while other permanent cell lines do. The viability of 3T3 cells in suspension remains unchanged for at least three days with respect to plating efficiency, vital staining and resumption of normal growth when transferred into monolayer culture. When monolayer 3T3 cells in G1 phase are suspended they remain in G1 phase. Cells already in S phase which are suspended complete ongoing DNA synthesis and mitosis and then are arrested in the G1 phase. Progress through the cell cycle is reinitiated after suspended cells attach to a surface. When monolayer cells in late G1 phase (just before entering S phase) are put in suspension cultures they do not initiate DNA synthesis.

A comparison of the distribution patterns of glycoproteins released into the medium by normal and oncogenic virus-transformed mammalian cells on 5% and 2–12% linear gradient sodium dodecylsulfate-polyacrylamide gels was made. Marked differences were observed in the distribution patterns obtained with the two types of gels. The gradient gels separate the glycoproteins into three discrete groups, while the 5% gels show only numerous small peaks. It appears that gradient gels may be of unique value in the separation of glycoproteins and may permit the identification of groups of compounds not distinguishable on single porosity gels.

It was previously shown that BHK21 cells were arrested in the G1 phase of the cell cycle when cultured in medium lacking serine. In this study the effect of serine limitation on protein synthesis was examined. Shifting cells from medium supplemented with 10% fetal calf serum to medium supplemented with 10% dialyzed serum resulted in a 50% reduction in the rate of protein synthesis. The reduced rate was attained within 4–10 min after shift-down and was restored completely within 5–15 min after shift-up to 10% dialyzed serum plus 0.05 mM serine, the same approximate concentration of serine present in 10% fetal calf serum. Exogenous serine appears to be incorporated into protein from a precursor pool which is functionally compartmentalized inasmuch as incorporation of serine into protein became linear within 10 min after the addition of label while the specific activity of serine in the acid soluble fraction did not attain a constant value during 60 min of labeling. The serine: leucine ratio in total cellular protein was determined from cells cultured in ten percent dialyzed serum plus 0.05 mM serine by amino acid analysis and was compared with the ratio of [ 3 H]serine and [ 14 C]leucine incorporated into protein. The results indicated that 50–60% of the serine utilized for protein synthesis under these conditions was derived from the medium while the other 40–50% was generated within the cell.

Rous-sarcoma transformed BHK cells can be continuously cultured in a medium containing Eagle's Minimal Essential Medium, iron and biotin. The rate of cell multiplication increased when serine, or serine plus other non-essential amino-acids were added to the medium. With biotin deleted from the medium there is a reduction in DNA synthesis and most cells are blocked in G1.

The relative rate of turnover of individual membrane proteins and glycoproteins in exponentially growing and contact-inhibited MK2 cells was investigated. Plasma membranes were isolated from cells that had been sequentially labelled with 14C and 3H isotopes of leucine and glucosamine. The membranes were then solubilized in sodium dodecylsulfate and their polypeptides separated by acrylamide gel electrophoresis. The 3H/14C ratios of the individual polypeptides reflected their relative rates of turnover. The proteins and glycoproteins of the exponentially growing cells exhibited markedly heterogeneous rates of turnover. In contrast, polypeptides in membranes of contact-inhibited cells exhibited a lesser degree of heterogeneity of turnover. In both exponential and contacted cell membranes a glycoprotein with a high apparent molecular weight exhibited the fastest rate of turnover.

Summary A water soluble fraction, elinin, was separated from human red cells. It contained the Rh, A and B factors when prepared from red cells of the respective type. Another fraction, S protein, was also separated from red cells and was devoid of the above-mentioned factors. The serological properties of these fractions and the stability of the Rh and A factors under various conditions are described.

Moskowitz, Merwin (Purdue University, Lafayette, Ind.). Separation and properties of a red cell sensitizing substance from streptococci. J. Bacteriol. 91: 2200–2204. 1966.—An antigen that binds onto red cells and causes them to be agglutinated by antiserum was separated from streptococci. Various procedures to extract the antigen from streptococci were investigated, and the greatest amount of antigen was obtained by extraction of cells with a phenol-water mixture. The reaction of the antigen with red cells was shown to be reversible by use of the Ashby mixed agglutination technique. The antigen also combines with a number of different tissues, and it was demonstrated that the antigen could be transferred from red cells to tissues and vice versa. An hypothesis is presented on the basis of these findings which suggests a possible role for this antigen in the etiology of rheumatic fever.

Summary1. Elinin, a lipoprotein fraction of the red cell exhibiting Rh activity has been isolated in relatively pure form largely by physical methods.2. Injection of elinin from Rh positive cells into animals has so far failed to yield a measurable titer of anti-Rh antibodies.3. Injection of elinin into human subjects who have been sensitized to the Rh factor by pregnancy or transfusion failed to increase the titer of Rh antibodies in the serum. Two of the subjects injected showed subsequent response to injection of whole Rh positive cells.4. Injection of 1 to 1.5 g of active elinin intramuscularly failed to effect a change in titer of circulating antibodies.

The toxicity of Clostridium perfringens was determined in mice utilizing three different routes of inoculation. There was marked variation in the minimum lethal dose with the different routes; the largest amount was required for the intracutaneous route, less for the intravenous route, and least with the intraperitoneal route. The relationship of toxicity to the lecithinase content of different toxin preparations was assayed. It was found that the toxicity of toxin preparations in different states of purification was not correlated with their lecithinase content.

Cells resistant to the action of relatively large quantities of streptomycin have been isolated from a streptomycin sensitive strain of mammalian cells. Sensitive and resistant clone cultures were also separated from the parent strain by cloning techniques in the absence of streptomycin. Streptomycin resistance and sensitivity is maintained after repeated subculturing in the absence of streptomycin. Some of the factors affecting the resistance of cells to streptomycin have been studied and it was found that the nutritional and physical environment of the growing culture affected the behavior of the cells in streptomycin. Streptomycin sensitive cells appear to release more 260 mμ absorbing material than streptomycin resistant cells.

The inhibition of Clostridium perfringens α-toxin by ethylenediaminetetraacetate (EDTA) and diethylenetriaminepentacetate (DTPA) was studied utilizing three different in vitro assay procedures: diffusion on egg yolk-agar, disintegration of muscle sections, and manometric assay with partially purified lecithin as substrate. DTPA was 10 to 20 times more efficient as an inhibitor than EDTA in systems containing relatively large amounts of calcium ; these observations were similar to those observed in previous in vivo protection studies. A number of other chelating agents were tested for their ability to inhibit α-toxin in vitro and protect mice against it; the chelating agents which were the most efficient in vitro inhibitors had the greatest in vivo protective ability.

The relative ability of the calcium chelates of calcium disodium ethylenediaminetetraacetate (EDTA) and calcium trisodium ethylenetriaminepentaacetate (DTPA) to protect mice against lethal doses of Clostridium perfringens α-toxin was investigated. Their protective ability was assayed by the increase in survival time of mice which had been given large doses of toxin, and by determining the median protective dose of chelate that would protect mice against toxin at a minimum lethal dose of two. In both assay procedures, intraperitoneal, intravenous, and intracutaneous injections of toxin were utilized, and with each toxin injection route the protective ability of the chelate was determined with the three routes of injection. DTPA was 10 to 20 times more effective than EDTA with both types of assay procedure and with all injection routes. DTPA may be superior to EDTA as a protective agent not only because it binds zinc to a greater extent, but also because of its greater retention in the body and its ability to gain entrance into cells. It appears that DTPA may be of value as a therapeutic agent in gas-gangrene.

Jackson, Robert W. (Purdue University, Lafayette, Ind.), and Merwin Moskowitz . Nature of a red cell sensitizing substance from streptococci. J. Bacteriol. 91: 2205–2209. 1966.—A method for purifying a streptococcal antigen which sensitizes red cells to agglutination by antiserum is described. The antigen, when purified by this method, is almost exclusively composed of glycerophosphate and d -alanine. The ratio of alanine to glycerophosphate varies from 1:5 to 1:3. The glycerophosphate is polymerized and is thus a teichoic acid. The polyglycerophosphate appears to be the antigenic determinant for agglutination. d -Alanine is readily removed by mild base and appears to be necessary for the attachment of the teichoic acid to red cells. Quantitative removal of alanine does not affect the ability of the polymer to absorb antibody from serum.

SummaryA method for separation of a fraction from horse serum which does not react in tests for protein and which is capable of substituting for whole serum in tissue culture medium has been described. When this fraction was used as a supplement in synthetic medium, cell growth was obtained which compared favorably with controls grown in serum supplemented medium.

The rate of protein degradation in 3T3, 3T3/41, SV40 virus-transformed 3T3, BHK21, Rous sarcoma virus-transformed BHK21 and MRC-5 cells was assayed in monolayer and suspension culture by pre-labeling the cells with radioactive leucine and measuring the loss of radioactivity from the cells. The rate of protein degradation of 3T3 and MRC-5 cells in suspension culture, under which condition they do not grow, was greater than that of other cell lines which are capable of growing in suspension. In all cell lines it was greater in suspension culture than in monolayer culture. There was an inverse correlation between the rate of protein degradation and the growth rate of the cells. The addition of insulin to 3T3 cells in suspension culture causes the reduction of the rate of protein degradation, increase of cellular protein and stimulation of DNA synthesis.

Materials released into culture medium by transformed and untransformed baby hamster kidney cells labelled with glucosamine, sulfate, fucose or leucine were characterized. Some of the components could also be labelled by iodination of intact cells, indicating their surface origin. Analysis on gradient polyacrylamide sodium lauryl sulfate gels demonstrated that a group of high apparent molecular weight glucosamine-labelled components were more abundant in materials released from Rous sarcoma virus-transformed baby hamster kidney cells than from baby hamster kidney cells or polymo virus-transformed baby hamster kidney cells. The relative rates of release of glucosamine-labelled components from transformed and untransformed cells were similar except that the transformed baby hamster kidney cells released some large molecular weight components slightly more rapidly than baby hamster kidney cells. Treatment of labelled medium materials with testicular hyaluronidase removed much glucosamine label from the materials but did not affect the amounts of other labels. After treatment with hyaluronidase, the patterns of labelled conditioned media from both transformed and untransformed baby hamster kidney cells were qualitatively and quantitatively very similar, suggesting that the differences seen in untreated labelled conditioned media were due to the presence of hyaluronidase-sensitive materials associated with medium materials rather than to actual differences in glycoproteins.

Biotin or a serum lipid extract stimulated proliferation of G1 arrested Rous sarcoma virus-transformed BHK cells in modified Eagle's MEM (BM). The cells could be maintained continuously in BM plus biotin (BMB), but not in BM plus serum lipid extract (BM · L). Avidin inhibited growth stimulation when added to BMB, but did not inhibit growth when added to BM · L. 14C-acetate incorporation into total cellular lipids was stimulated in BMB, but not in BM. Thin-layer chromatography of the labeled cellular lipid extract indicated that relatively large amounts of 14C-acetate were incorporated into phosphatidylserine and little into the other major phospholipids. In the neutral lipids, the largest amount of incorporation was in cholesterol. G1 arrested cells multiplied rapidly in BM supplemented with dialyzed serum (BM · DS), but they did not multiply in BM with delipidized serum (BM · DLS). The addition of biotin or serum lipid extract to BM · DLS stimulated growth. Growth stimulation in BM · DLS by biotin was inhibited by avidin, but avidin had no effect on growth stimulation by serum lipid extract. Biotin stimulated additional multiplication in BM · DS and avidin inhibited this additional growth stimulation. These results suggest that growth stimulation requires lipids supplied by serum lipids or by de novo synthesis stimulated by biotin. In the absence of serum, the stimulation of the synthesis of growth factor(s) by biotin are also required for continuous multiplication.

A dialysate of fetal bovine serum stimulated the growth of BHK cells in a medium containing 1% dialyzed serum. The growth-stimulating activity of the dialysate was separated in a single peak with Biogel P-2 chromatography, and a single active component was further fractionated with paper and thin-layer chromatography. The component was identified as biotin. Pure biotin behaved the same as the active component in the dialysate and had the same growth-stimulating activity. The dialysate contained only the free biotin that was present in serum. Free biotin was found in relatively large amounts in fetal (bovine and human) sera, and only a very small percentage of dialysates of adult (bovine, horse, swine, and human) sera had traces of growth-stimulating activity. The possible role of free biotin in the body is discussed.

There is a low uptake of leucine in suspension cultures of 3T3 cells relative to the uptake in sparse monolayer cultures. The pattern of uptake of deoxyglucose is similar in suspension and monolayer cultures and changing the medium or adding serum stimulates uptake under both culture conditions. The uptake of uridine and adenosine is greater in suspension culture than in monolayer culture. Cells do not multiply in suspension culture but do multiply in monolayer culture and thus there is a correlation between uptake of leucine and conditions which stimulate cell multiplication, but not correlation of the uptake of deoxyglucose, uridine and adenosine with these conditions.

3T3 cells in subconfluent culture take up leucine through a transport system which has a relatively high affinity for leucine (M system). When the culture becomes confluent, the M system is turned off and leucine is transported by another system which has a low affinity for leucine (S system). The M system is reactivated by transferring the cells into subconfluent cultures. In suspension cultures 3T3 cells, initiated from confluent cultures, the M system is not activated and leucine is transported by the S system. In cells suspended from subconfluent culture, the M system continues to operate at a high level for four hours and then is gradually turned off. Tumor virus transformed 3T3 cells (SV3T3 and Py3T3) grow quite well in suspension culture and transport leucine both in monolayer and suspension through a high affinity system, with a high Vmax value. A derivative of 3T3, 3T3/41, which grows in suspension much more slowly than tumor virus transformed 3T3 cells, also takes up leucine through a high affinity transport system both in monolayer and suspension but its Vmax value is lower than that of the transformed cells.

SummaryIt has been demonstrated that CaEDTA can protect mice against a lethal dose of Cl. perjringens toxin, and guinea pigs against experimental gas gangrene.

Cultured mammalian cells killed by streptomycin were essentially unaffected by an identical concentration of dihydrostreptomycin.

IN the course of a study on factors that may be involved in the in vivo destruction of stored red cells, an experiment was performed which suggests that the antigenicity of red cells is altered when blood is kept for a long period of time under standard storage conditions, and individuals may be sensitized with such stored red cells.

A low molecular weight fraction was isolated by gel filtration from commercially prepared tissue hydrolysates. These low molecular weight materials could replace the serum supplement for the growth of some established cell lines in vitro , and they enabled the cultured cells to grow at a rate comparable to that observed with the serum supplement. It would appear that the active material is a mixture of products, possibly peptide in nature, with molecular weight between 700 to 1600. A high molecular weight fraction separated from these hydrolysates did not stimulate the growth of the cells to the same extent and, under some conditions, caused the cells to clump.

SummaryA simple method for isolating single tissue culture cells has been developed. The cells are isolated on small glass squares which can be transferred to any desired culture vessel or testing apparatus. This procedure has been used for the preparation of clone cultures.

Growth promoting and growth inhibiting factors were separated from an extract of porcine pancreas by dialysis. The growth promoting fraction in the dialysate initially had a slight toxic effect on the cells, but upon recovery they grew to a greater extent than in serum supplemented medium. The addition of human serum albumin to dialysate supplemented medium protected the cells from initial toxic effects and growth in this medium was as much as three times greater than in serum supplemented medium.

IT was reported previously that ethylenediamine tetraacetic acid was capable of protecting mice against the toxin of Clostridium perfringens 1. The protection thus afforded was shown to be probably due to the removal of a metallic ion or ions necessary for the activity of the toxin from the area where the toxin was acting. Inasmuch as the metallic ion or ions necessary for the action of the toxin apparently were higher in the preferential chelation series than calcium, the calcium chelate was shown to be able to protect when given systemically2. In this report, studies on the duration of the protection afforded by calcium ethylenediamine tetraacetic acid are presented and their implications are discussed.

IN the course of a study of the effects of formalin on red cells it was noted that formalin was capable of completely inhibiting the agglutinability of red cells by antisera without destroying the antigens of the cells.