Ruth K. Silver

Erythropoietic kinetics in sheep studied by means of induced changes in hemoglobin phenotype

This investigation is concerned with the kinetics of the reciprocal relationship between sheep hemoglobin (Hb) A and Hb C formation in response to anemia. The relative synthesis of the hemoglobin types was assessed at various times in bone marrow erythroid cells incubated in vitro with (59)Fe. The changeover from Hb A to Hb C formation lagged by about 3 days behind the development of anemia and was complete within about 11 days. After recovery from anemia the reciprocal change back to preanemic conditions proceeded at a much slower rate, Hb C formation gradually declining to unmeasurable levels over about 25 days. Infusions of plasma with high erythropoietin titre induced the formation of relatively large quantities of Hb C in erythroid cells of nonanemic sheep, demonstrating the central importance of a humoral mechanism in the change of expression of the hemoglobin genes. THE FOLLOWING CONCLUSIONS WERE DRAWN: hemoglobin phenotype is determined at a stem cell level. Erythroid stem cells appear to undergo gradual renewal. The identity of the plasma factor which induces Hb C formation is not yet known; it is not present in plasma from nonanemic sheep, and its production is not dependent upon hemoglobin genotype. If the plasma factor turns out to be erythropoietin, then this hormone must have an important influence on the pool of erythroid stem cells.

PRESERVATION OF CELL CULTURES BY FREEZING IN LIQUID NITROGEN VAPOR.

Summary Nine continuous, 2 diploid and one primary cell line were successfully preserved by freezing in liquid nitrogen vapor at cooling rates of 1.4°C/min to 43°C/min, but poor survival was observed when cells were frozen by immersion directly in liquid nitrogen. The cells were frozen directly in a liquid nitrogen refrigerator by placing the ampuls at different distances below the lip, demonstrating that elaborate and expensive cell preserving equipment is not required. The plating efficiency technique was shown to be the most sensitive indicator of cell damage.

Historical development of cell and tissue culture freezing

Abstract In conclusion, the use of glycerol or dimethyl sulfoxide has permitted successful freezing, storage, and thawing of most, but not all, animal cell cultures. Storage in liquid nitrogen without detectable change in observable characteristics appears to be indefinite. Methods are empiric, understanding of the basic physics and chemistry involved are fragmentary, and results cannot always be exactly duplicated within one laboratory or in different laboratories. These problems will be solved in the future by the combined application of many disciplines with overlapping interests in molecular biology. In the meantime, present methods permit indefinite cold storage of most cell cultures with a great saving in labor and inconvenience, while avoiding accidental loss, contamination, or mutation of the cells during repeated serial passage.

The Formation of Foetal and Adult Haemoglobin in Cell Cultures of Neonatal Calf Marrow

Summary Neonatal erythroid cells in the bovine respond to erythropoietin in vitro with a pari passu increase in the synthesis of both foetal and adult haemoglobins. With marked stimulation the effect upon the latter predominates. The conditions of cell culture, however, promote the formation of a protein with chromatographic characteristics of foetal gamma‐globin subunits, quite independently of the presence of erythropoietin or of haem synthesis. The results suggest that erythropoietin might be assigned a significant although indirect role in the gradual switchover of haemoglobin types during development.

Studies on the Enzymatic Properties of Influenza Viruses, I. The Action of Influenza B Virus and RDE on the Hemagglutinin Inhibitor of Human Meconium.

Abstract In purified preparations of human meconium, inhibitors of hemagglutination by influenza viruses have been described which are destroyed upon incubation with active virus. We have attempted to identify chemically the products liberated during such interaction. A solution of purified meconium of high hemagglutinin inhibitory titer (HAI) was incubated with strain Lee of influenza B virus. HAI was abolished overnight, no drop in HAI occurred in meconium solutions incubated with uninfected allantoic fluid or with virus previously rendered noninfective by heating at 56°. Dialyzates from meconium treated with active virus gave a violet color with Bials orcinol reagent; dialyzates from control preparations were Bial-negative. From the Bial-positive dialyzates an acid was obtained in crystalline form which, on the basis of its X-ray powder diffraction diagram, melting point, optical rotation, and infrared spectrum was found to be identical with N-acetylneuraminic acid, as isolated by mild acid hydrolysis from the hemagglutinin inhibiting mucoproteins of sheep submaxillary mucin, human milk, and meconium. Thirteen per cent of the total neuraminic acid present in meconium was consistently liberated by the virus; a preparation of receptor-destroying enzyme (RDE) was more active and liberated as much as 48% of the neuraminic acid of meconium. In addition, dialyzates from virus or RDE-treated meconium contained a still unidentified ninhydrin-positive substance absent from control dialyzates.

VIABILITIES OF FROZEN MAMMALIAN CELLS FOLLOWING SLOW THAWING.

Summary 1. Under the conditions described, good viability of frozen mammalian cell cultures was often obtained following slow thawing in the presence of added di-methylsulfoxide. 2. Variation of pH of the freeze medium had no effect on viabilities of frozen HeLa, SJ. cells following slow or rapid thawing. 3. Dimethylsulfoxide is superior to glycerol in preventing damage of the cells tested during freezing. This is particularly evident when slow thawing is employed.

A PREMIXED POWDER FOR PREPARATION OF TISSUE CULTURE MEDIA.

Summary Five different tissue culture media prepared as a dry powder by blending the ingredients in a ball mill followed by desiccation were reconstituted and evaluated for their ability to support cell growth of ten different tissue culture cell lines. Eagles BME, Eagles MEM, Medium 199, NCTC 127 and NCTC 109 supported good cell multiplication as compared to the control Eagles MEM prepared from 10 × or 100X concentrated liquid stock solutions. Plating efficiencies of 8 of 10 cell lines were higher in media prepared from the powdered Eagles MEM than in the control media, Plating efficiency data were confirmed by growth curves in roller tubes and by growth of massive inocula in milk dilution bottles. Additional important advantages of using dry powdered culture media include: low cost of powdered media as compared to the present commercial liquid concentrates; greater stability; greater uniformity of the chemical ingredients because of the ball milling process and the rigid selection of large bulk lots of basic ingredients; low shipping charges; and ease of storage for long term experiments. The authors wish to thank Drs. Richard C. Meyer and Robert E. Stevenson, Nat. Inst. Health, and Herbert R. Morgan, Univ. of Rochester Med. School, N. Y., whose efforts as members of the Subcommittee on Media and Substrates of the Cell Culture Collection Committee initiated this study. Thanks are also due Dr. Leonard Hayflick, Wistar Inst., for helpful advice, and Mr. Michael Peluse and Mrs. Elizabeth Kauderer for technical assistance.

Use of Dielectric Heating (Shortwave Diathermy) in Thawing Frozen Suspensions of Tissue Culture Cells.

Summary 1. Thawing of frozen mammalian cells by well controlled shortwave diathermy is feasible. 2. Such thawing causes no apparent injury to the cells. 3. Shortwave diathermy offers no advantage over agitation in a 37°C water bath for thawing small masses of frozen tissues and cells. 4. The use of shortwave diathermy appears to be desirable for thawing large masses of tissue such as the dog or human kidney and the present studies indicate that this method can be safely used.

THE CONTROL OF HEMOGLOBIN PHENOTYPE IN CALVES AND SHEEP

The genes for nonalpha globin chain formation undergo sequential changes in activation during development. In man, the 0-chain of adult life ultimately replaces the tand y-chains of earlier developmental stages. Sickle cell anemia and Cooley’s anemia, the two most severe hemoglobinopathies, are both diseases of the phenotypic expression of the abnormal adult gene for beta globin chain expression. The possibility that gene expression may be altered in such a way as to suppress the defective gene while reactivating a normally silent but nondefective gene is considered in the studies that follow. These studies were conducted in sheep in which a normally silent gene for adult nonalpha globin chain production becomes active during anemia. Newborn calves were also used as a model for studies of fetal hemoglobin synthesis. Hemoglobin polymorphism is present in sheep. The two prevalent adult hemoglobin types are called A and B. Sheep possessing the gene for H b A predictably undergo a change in gene activation in response to anemia; the H b A is replaced by a new hemoglobin called C. This new hemoglobin differs from that which it replaces only in the @-chain. Thus, there appears to be a reciprocal response of two closely related @-chain genes during anemia. The switchover may be complete within ten days of the onset of severe anemia.’” The analogy between this change and the reciprocal change during development from yto @-chain formation is obvious. In the former instance, however, the change is reversible, whereas in the latter it is irreversible. The results to be presented will show: ( 1 ) that the stimulus of severe anemia produces consequences in the erythroid stem cells which persist for about 25 days beyond the cessation of the stimulus, and (2) that a factor present in plasma taken from anemic sheep is capable of producing the change in hemoglobin synthesis in nonanemic sheep. All the evidence to date is consistent with the hypothesis that this factor is the same as erythropoietin.