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SummaryExposure of hamster and mouse embryo cells to X-irradiation prior to infection with Simian virus 40 (SV40) significantly increased the frequency of transformation in vitro. Neonatal hamsters were tested for increased sensitivity to SV40 tumorigenicity after exposure to localized, low-level X-ray at the site of virus inoculation in vivo. Results showed that irradiation of a target area prior to infection markedly decreased the time to tumor appearance and increased the frequency of tumor occurrence. No tumors occurred in irradiated animals that did not receive virus. Although low doses of localized X-ray may have resulted in modest immunologic suppression, this was not a primary factor in tumor enhancement. Tumors appearing in SV40-infected animals uniformly possessed viral neoantigens.

WE wish to describe new serological techniques based on changes in buoyant density occurring when antibodies, or antigens, or both, are attached to latex beads. For the density change to be observed, the mass of the attached particles must be an appreciable fraction of the total mass. Using very small carrier beads less than one hundred antibody molecules or one virus particle per bead can be detected. The speed and sensitivity of the method make it directly applicable to the clinically important problem of rapid identification of infectious agents.

Zonal separation of particles in density gradients contained in hollow or tubeless rotors has been explored with the aim of developing a preparative counterpart of the analytical ultracentrifuge. A rotor with a capacity of 1625 milliliters has been tested up to 22,500 revolutions per minute, with sucrose density gradients used. Excellent separation of subcellular particles has been achieved as well as partial separation of the albumin and globulin peaks of serum.

The instrumentation requirements for agricultural research in the 21st century hinge on whether or not complete analyses of animal and plant cells will be required, on the cost of performing the analyses, and on the cost and complexity of the analytical instrumentation needed. It is emphasized that nature’s response to modern agriculture is to evolve pests and diseases better fitted to attack new genetically uniform plants, or to escape pesticides or herbicides. For all future history, new strains of crop plants, new herbicides and pesticides, and new animal strains will be required periodically. Existing native plant varieties now provide genes for resistance which are empirically discovered, and bred into high-yield strains by conventional methods. It is suggested that this process will not be endlessly successful, and that at some future time the required genes will not be found. Then, true genetic engineering with rational design and optimization will be required. This means the complete sequencing of plant and animal genomes, the identification and characterization of all gene products, computer modelling of cell function, and ultimately the introduction of sets of modified or totally synthetic genes. We review the status of some of the instruments required, and suggest future directions for their development.

Complex manifolds are required to arrange and control flow of liquids during the loading and unloading of zonal centrifuges, in vaccine production using the continuous-sample-flow-with-banding K-type centrifuge, in rapid recycling affinity chromatography, in continuous flow analyzers, and in a variety of devices and systems using density gradients. In many instances permanent metal manifolds or valves are to be avoided because they are expensive to fabricate, are difficult to clean, and may accumulate sediments and microorganisms which are not observed because they are opaque. Glass manifolds are fragile and are not easily made by the uninitiated. Connections between plastic tubing and glass may separate under pressure, especially when detergents are present. The advantages of using plastic tubing, and hemostats in place of valves to control complex flow patterns, have been previously stressed. A simple solution to the problems of manifolds of metal or glass is to use various sizes of Tygon tubing cemented together with cyclohexanone containing approximately 5 to 10% dissolved Tygon. Very small tubing (less than 1/8-in. i.d.) may be clogged by the cement and should be cleared of liquid cement with low-pressure air. Very large pieces of Tygon tubing may need to be held in place with clampsmore » or jigs until the cement has set. These Tygon manifolds have been found very resistant to tearing and easily cleaned and replaced.« less

The two-dimensional electrophoretic technique of O'Farrell has been adapted to the analysis of human plasma proteins, and 30 polypeptides have been identified in the pattern produced. Genetic variants involving charge (isoelectric point) or size (molecular weight in the presence of sodium dodecyl sulfate) changes should be routinely detectable in at least 20 proteins at once, facilitating studies of human mutation rates.

Publisher Summary This chapter focuses on the usage of the large-scale two-dimensional ISODALT gel electrophoresis system in immunology. At Argonne National Laboratory, the ISODALT system, where ISO describes the charge separation and DALT stands for size separation, is a large, multipurpose operation. The chapter also describes one version of the ISODALT system adapted at the Basel Institute for Immunology for specific problems occurring in immunology. In 1975, O’Farrell developed an analytical technique for the resolution of complex protein mixture using a combination of two separation procedures—charge separation and size separation. The electrophoretograms obtained by this technique allow a fine comparison of the protein pattern within a single run. The final aim is to perform a comparison using a computer, the function of which is not only to compare two gels adjusted by matching, stretching, and fitting but also to analyze gels by comparing the presence or absence of individual spots or constellations of spots with a cumulative experimental data file.

The location, molecular structure, and stage of assembly of viruses and viral components in cells, tissues, and body fluids at various times after infection are important for the understanding of viral diseases. It is equally important to examine in detail the morphological, structural, and molecular alterations occurring in native cell constituents after viral infection.

The ISO-DALT system of two-dimensional electrophoresis was developed as a series of modifications of the original technique of O'Farrell. Since the appearance of the first two open literature publications describing the ISO-DALT system in 1978, the technique has continually been refined. As a supplement to these first references, and to those that have followed, we have written a series of recipes and more detailed laboratory procedures that incorporate refinements and tricks of the trade as they have developed during our use of the system. The present collection is the 6th version in this constantly evolving series, and represents the state of art as of October, 1981. This, the sixth edition, has been prepared in order to bring up to date the changes that we have found helpful, as well as to add a few new details. (ERB)

PACKED potato starch, which has been widely used for zone electrophoresis, offers certain advantages when employed as a supporting medium in the ultracentrifuge. Boundaries are stabilized, making recovery of fractions easier, and the proteins thus far studied sediment at least twice as rapidly in starch as in free solution. This has been shown in experiments with rat haemoglobin (1 per cent) or bovine serum albumin (1–5 per cent) stained with bromphenol blue centrifuged in starch and in free solution under identical conditions. Centrifugation was done in the ‘Spinco’ Model L centrifuge by use of an SW 391 swinging-bucket rotor at approximately 20°. A 0.1 ionic strength pH 7.5 phosphate–sodium chloride buffer was used in each instance. The ‘Lusteroid’ centrifuge tubes were completely filled with a slurry of starch–protein mixture and accelerated briefly to 20,000 r.p.m. After deceleration, excess fluid was removed and more starch slurry added. The packing procedure was repeated a second time and excess fluid again removed. It is important that the starch suspensions be freshly made, since part of the starch appears to dissolve slowly with time, destroying the effect observed here.

Membranous material was prepared from Ehrlich ascites carcinoma cells by zonal centrifugation. The distribution of fatty acids among the individual neutral lipid and phospholipid classes isolated from the membrane preparation was determined. Survey electron microscopy indicated that the preparations consisted of vesicles of rough surfaced endoplasmic reticulum, lysosomal-like structures, virus-like particles, few intact mitochondria, and unidentified amorphous material, in addition to plasma membranes. Lipids and proteins accounted for approximately one-third and two-thirds of the membranous material. Phospholipids accounted for two-thirds of the total lipids. The membranous material contained higher levels of free fatty acids, cholesterol, diacyl phosphatidyl choline, diacyl phosphatidyl ethanolamine, and lower levels of glyceryl ether diesters and cholesterol esters than whole cells. The fatty acid composition of all the lipid classes isolated from the membranous material, except phosphatidyl inositol and cholesterol esters, was similar to the fatty acid distribution of the corresponding classes obtained from whole cells. Cholesterol esters contained elevated levels of 16:1 and stearic acid accounted for 88% of phosphatidyl inositol fatty acids. Phosphatidyl choline and phosphatidyl ethanolamine isolated from the membranous preparations contained 1% or less alkyl acyl phosphatides, and plasmalogens were not detected. The lipids of whole Ehrlich ascites cells have been shown to contain high levels of ether-linked lipids, whereas these data indicate that alkyl and alk-1-enyl glyceryl ethers are virtually absent from membrane preparations of these cells. These results indicate that the ether-linked lipids are not distributed equally among all cell structures.

Plasma membranes of rat liver were isolated by zonal centrifugation for analysis of lipid classes and fatty acid composition. The yield of plama membranes by this technique was 880 pg of protein and 390 pg of total lipid per g of liver. Neutral lipids represented 27% of the total extractable lipids, and this was mainly cholesterol. Phosphatidylcholine accounted for almost 40 % of the phospholipids in the plasma membranes of rat liver. Phosphatidylethanolamine and sphingomyelin amounted to approximately 20 and 18 %, respectively, of the total phospholipids. Phosphatidylserine and phosphatidylinositol were not resolved from each other; this mixed fraction contained approximately 13 % of the total lipid phosphorus. Lysophosphatidylcholine and polyglycerolphosphatides plus phosphatidic acid were present as minor components of the membrane lipids. An unidentified polar lipid containing no phosphorus was resolved from other phospholipids and accounted for approximately 12% of the total lipids. The alkyl and alk-1-enyl ethers of glycerol liberated from the total lipids of liver plasma membranes were 0.97 and 0.24%, respectively. The fatty acid composition of P recise knowledge of the composition of mammalian cell membranes has been limited because of the difficulty in preparing pure material. Until recently our understanding of the composition of cell membranes was limited to data concerning the erythrocyte (Ponder, 1961 ; De Gier and Van Deenen, 1961), which might be a poor model for mammalian cells. But in 1960, Neville described a procedure for isolating rat liver cell membranes, and several other investigators (Emmelot et a/., 1964; Takeuchi and Terayama, 1965; Ashworth and Green, 1966) partially characterized the lipids found in such membrane preparations. Recently, Skipski and coworkers (1965), using the isolation procedure of Ne* From the Lipid Research Laboratories, Medical Division of the Oak Ridge Associated Universities, Oak Ridge, Tennessee, under contract with the U. S. Atomic Energy Commission and with the Joint National Institutes of Health, Atomic Energy Commission Molecular Anatomy Program (the Molecular Anatomy Program is sponsored by the U. S. Atomic Energy Commission, the National Cancer Institute, the National Institute of General Medical Sciences, and the National Institute for Allergy and Infectious Diseases), Oak Ridge National Laboratory (operated for the U. S. Atomic Energy Commission by the Nuclear Division of Union Carbide Corp.), Oak Ridge, Tennessee 37830. Receioed April 4, 1968. t Present address: Lovelace Foundation for Medical Education and Research, Albuquerque, N. M. 87108. 2826 all phospholipids, triglycerides, and free fatty acids was determined by gas-liquid partition chromatography. The fatty acid composition of phosphatidylcholine and phosphatidylethanolamine was similar. About twice as much palmitate (28%) and stearate (31 %) as linoleate (14x) and arachidonate (14%) was found in these fractions. The fatty acid composition of the mixture of phosphatidic acid and polyglycerolphosphatides was similar to that of phosphatidylcholine and phosphatidylethanolamine. The sphingomyelin fraction of the liver cell membranes contained most of the polyunsaturated acids (Go and above), whereas lysophosphatidylcholine contained the highest percentage of saturated fatty acids. The unidentified polar lipid containing no phosphorus had a fatty acid composition of 16:O (32 %), 18 :O (48 %), 18:l (lo%), and 18:2 (10%). Triglycerides were comprised mainly of 16 :0, 18 :1, and 18 :2 acyl moieties. The plasma membranes of rat liver isolated by zonal centrifugation appear to have many features of lipid composition similar to those reported by others for pure membranes of rat erythrocytes. ville, presented a detailed analysis of lipid classes in plasma membranes of rat liver. However, the plasma membranes isolated by the technique of Neville (1960) and the modifications of Emmelot et al. (1964) and Finean and coworkers (1966) were shown by Finean er ai. to yield a preparation contaminated (10-15 %) with mitochondria and endoplasmic reticulum. A later report by Skipski’s group (Barclay et ai., 1967) also indicated that the membrane fraction contained swollen mitochondria with fragmented cristae, amorphous material, and small vesicles of unknown origin. Liver cell plasma membranes essentially free of other particulate constituents were recently sedimented (ElAaser et ai., 1966) in the A-XI1 low-speed zonal centrifuge rotor (Anderson et al., 1966b) as a band that occupied an intermediate position between nuclei and mitochondria, confirming the view that cell-membrane fragments tend to sediment faster than mitochondria. Anderson et al. (1967, 1968a) have extended the zonal technique to achieve sequentially a high-resolution ratezonal separation and an isopycnic-zonal separation of pure liver cell membranes in the A-XI1 rotor. This paper describes the lipid classes and fatty acid composition of each lipid class found in the plasma membranes purified by zonal centrifugation. It also indicates the value of zonal rotor systems which can be partially unloaded P F L E G E R , A N D E R S O N , A N D S N Y D E R D ow nl oa de d by U N IV O F V IC T O R IA o n O ct ob er 1 9, 2 00 9 | h ttp :// pu bs .a cs .o rg P ub lic at io n D at e: A ug us t 1 , 1 96 8 | d oi : 1 0. 10 21 /b i0 08 48 a0 19 VOL. 7, N O . 8, A U G U S T 1 9 6 8 during a rate separation and therefore allow one fraction to be banded isopycnically in the same rotor. The advantage of unloading from the rotor edge is apparent; new experimental rotors (B-XXIII and B-XXIX) have been designed which may be unloaded from either the center or the edge. Experimental Procedures Isolation of Rat Liwr Plasma Membranes by Zonal Centrvigation. Livers from a total of 10 male SpragueDawley rats weighing approximately 300 g and maintained on a Purina Laboratory diet were used to prepare 13 different liver-membrane samples. Fresh unperfused rat liver was diced with scissors in chilled 1 mM NaHC03 (pH adjusted to 7.6 with NaOH) and homogenized with 15 even strokes of a loose-fitting Dounce homogenizer. The homogenate was filtered through eight layers of cheesecloth and diluted to a final volume of 30 ml/g of liver. The suspension was centrifuged for 15 min at 260 rpm (1720g) at 0" in an International PR-2 centrifuge using 250-ml tubes in the no. 259 head. The supernatant was discarded and the loose pellet was rehomogenized (five strokes) and resuspended in the bicarbonate buffer (12 ml, 1 mM NaHCOa/g of liver). This suspension was used for zonal centrifugation. The B-XV titanium zonal rotor recently described (Anderson et ul., 1968b) has a capacity of 1666 ml. It was operated in a modified Spinco Model L centrifuge equipped with an .fw2dt integrator previously described (Anderson et al., 1966a) and a device for sensing and controlling temperature. A 190-ml membrane suspension was layered over a 500-ml sucrose gradient (19-35 w/w % sucrose) formed by a Spinco Model 131 gradient pump. The membrane suspension was followed by an overlay (200 ml, 1 KIM NaHC03) and centrifuged at 5000 rpm until w 2 t = 2 X IO*. The first 800 ml (20 40ml fractions) at the top of the gradient was unloaded by displacing the lighter portion by a 45 % sucrose pushout solution, which was the same concentration as the cushion. After removal of the centripetal 800 ml, containing the bulk of the soluble, mitochondrial, and microsomal fractions (Figure l), a 50-ml HzO overlay was added and the rotor was accelerated to 20,000 rpm and run until w 2 f = 3.5 X lo9 at unloading. The membrane fragments were found to be sharply banded at an average of 41.04% (w/w) sucrose. The 260-mp optical density profile of Figure 1 shows the sharp banding of the membrane fraction (tubes 22-26). Observations of the banded membranes in numerous phase-contrast and electron micrographs (Figure 2) confirmed the high purity of the preparation. Other criteria used to judge the purity for the plasma membranes were (1) absence of cytochrome oxidase (D. H. Brown, personal communication), (2) high concentration of alkaline ATPase (Lieberman et al., 1967; Anderson et al., 1968a), and (3) absence of material banding above 42% sucrose during isopycnic centrifugation (Figure 1). Each of the membrane fractions was diluted 2:l (v/v) with bicarbonate buffer and centrifuged at 30,000 rpm for 30 min at 0" in a no. 30 rotor. The sucrose was drawn off and dis50

Liquid density gradients and experimental sample bands have been successfully recovered from a hollow-bowl centrifuge rotor in which a static gradient is reoriented into a radial configuration during acceleration and is subsequently recovered in its original orientation at rest. Analysis of fluid movements in the rotor indicate that very little shearing occurs during gradient reorientation. The reorienting gradient rotor principle makes feasible the construction of high-speed, large-volume rotors suitable for banding nucleic acids and proteins in salt gradients, and for the separation of macromolecules on the basis of their sedimentation rate through density gradients.

In the past two years, in collaboration with several colleagues, we have demonstrated the presence of antigenic determinants at the surface of SV40, adenovirus, polyoma tumors of hamsters, spontaneous and chemically induced tumors of mice (1–2) which are transiently present in fetal tissues but absent from adult tissues. In other laboratories chemically induced mouse tumors (3) and Maloney sarcoma tumor cells (4) also have demonstrable fetal or phase specific autoantigens (PSAA). The prospect has arisen that many and perhaps all cancers share antigens commonly present on the developing fetal membranes and that retrogenic phasing may be essential to the malignant process. Several major forms of human cancer have embryonic constituents reexpressed as exemplified by the alphafeto-protein of hepatomas or the carcinoembryomic antigen of cancers of endodermal origin.

The tissue distribution of enzymatic activities in intestinal metaplasia stomachs exhibiting chronic gastritis was compared histochemically with that of the small intestine in man.

A method is designed for the rapid fractionation of large volumes of suspensions of particles varying in size and density by gradient differential centrifugation. A centrifuge distribution head is used that permits the rapid construction of continuous liquid density gradients since the gradients are stabilized by centrifugal force as they are formed. The application of the method to the fractionation of rat liver breis is described.

VIRAL material used to prepare inactivated virus vaccines must be concentrated to provide sufficient antigenic mass to stimulate the vaccine's protective mechanisms, and purified to remove nonviral host material which may cause side reactions and sensitization to host (for example, egg) components. A continuous-flow zonal centrifuge, the K-II-B1,2, has been designed for use in large scale production of influenza vaccines. We have grown influenza virus in tissue culture and have concentrated and purified it in a single step process using this centrifuge.

A variety of structures, many of which resemble components of liver cells, have been shown to form in clear ultracentrifuged extracts of rat liver. The structures appear after dialysis against isotonic sucrose, pH 7.5 phosphate buffers, or after standing in the cold. When the pH is lowered to 5 or 6, fibrils are readily formed. It is suggested that these structures may form in rat liver breis. The possibility is discussed that cell components exist in equilibrium with their constituent molecules in solution and that mechanisms similar in part to those involved in structure formation in solution may also obtain in the living cell.

A method is described for determining total serum protein using a new, rapid microanalytic system in which quantification depends upon the color developed during the first few seconds of the biuret reaction. The method is in excellent agreement with the Kjeldahl method of nitrogen analysis for determining serum protein.

One of the aims of the Molecular Anatomy Program at Oak Ridge is the isolation in quantity of cancer associated antigens and antibodies. Proteins produced in cancer may be new to the host and thus be antigenic; or they may be antigenic because the immunological response apparatus of the host normally does not see them; or finally, if the proteins were produced before and reappeared, the immunological apparatus of the host may have been functioning poorly at the time or may have forgotten that it once saw them. In any case the proteins are antigenic to a proper challenge in an experimental animal.

Summary The K-II continuous-sample-flow-with-banding-9 zonal centrifuge hasbeen developed for large-scale virus isolation. The cylindrical aluminum rotor (capacity 3600 ml) contains a 3 liter gradient and has a 700 ml stream volume. Fluid line seals are located on opposite ends of the rotor, eliminating the possibility of cross-leakage. An air turbine drive is used to accelerate the rotor to 35,000 rpm (83,440g, maximum). The development of safe armor and control systems is described.

SummaryThe injection of heparin (50 mg intravenous) in the human causes a sudden fall in surface tension and a clearing of the lipemic plasma. The effect is believed to be due to the formation of a surface active heparin-phospholipid complex. The activity of the complex may be studied in vitro by mixing lipemic plasma with non-turbid plasma obtained after heparin injection. A gradual clearing is observed. The formation of a surface active heparin-egg lecithin complex has been demonstrated. Certain relations of these findings to pathological conditions are discussed.

Subcellular particles of homogenates of mouse spleen were separated at various centrifugal forces between 5000·106 and 30 000·106ω2t in the zonal centrifuge. Each fraction was analyzed for transplantation antigen, ATPase, nonspecific esterase and alkaline phosphatase. Under appropriate conditions, zonal centrifugation proved to be an efficient procedure to seperate particles containing high concentrations of transplantation antigen. After a centrifugal force of 5000·106ω2t, the antigenic particles were located in the 10–12% sucrose region, but they sedimented into denser sucrose particles anecame more disperse when subjected to larger total centrifugal forces. The density of teh particels in CsCl was about 1.2g/cm3. Transplantation antigen was solubilized by autolysis; the antigen was eluted with exclusion volume during gel filtration on Sephadex G-200.

Publisher Summary The chapter examines the theory that cancer is a disease of the mechanism of differentiation and that the compositional and behavioral changes observed in the majority of human tumors are due to the changes in the programming of normal genes. The basic problem is approached in four ways: first, by inquiring into the compositional changes that accompany malignant transformation; second, by investigating the available information on the differentiation program itself; third, if only normal substances (embryo or phase specific) and functions appear in most human malignancy, then by enquiring the way these are dealt with when they appear normally; and fourth, by posing whether the present knowledge of the complexity of the mammalian genome and of mechanisms for the control of gene expression is compatible with the basic thesis. The chapter explains the transformation-associated cellular alterations and the organization of differentiation from a descriptive viewpoint, with the question of whether the remnants of differentiation so described persist in tumor cells. A review of the major characteristics of cancer cells suggests that all essential characteristics for neoplasia are found in embryonic cells and that the selective reexpression of some of the genes involved in early development occurs in cancer.

VIRUS infection is widespread throughout the animal and plant kingdoms, and produces most human illness1, possibly including certain types of cancer2. Particles with viral morphology are easily isolated from sea3 or river water suggesting that they are ubiquitous. In this communication we have assembled evidence to support the view that viral transduction is a key mechanism for transporting segments of DNA across species and phylum barriers, and that evolution depends largely on this transfer. The evidence may be summarized as follows.

IMMUNIZATION of adult hamsters with irradiated foetal hamster or mouse cells produces strong immunity to SV40 cell challenge and simultaneously induces cytostatic (or C) antibody formation1. Furthermore, adenovirus 31 tumorigenesis can be interrupted by a single injection of hamster foetal cells if administered to neonatally infected hamsters 3 weeks after birth and before the appearance of the tumour2. SV40 viral oncogenesis could be similarly interrupted in male but not female hamsters2. This work provides support for the view that foetal and tumour cells share common antigens and that oncogenesis involves retrogenic activation of early foetal antigen genes in adult cells3,4.

THE improvement of methods for isolating cell nuclei requires the establishment of suitable criteria of nuclear intactness which may be used to evaluate changes in technique1. The following experiments were performed in the course of an effort to devise methods for following changes in the degree of polymerization of deoxyribonucleic acid during nuclear isolation.

Loss of viability, most often expressed as a failure of irradiated cells to divide indefinitely, has been the major criterion in evaluating radiation damage at the cellular level. In terms of the cell cycle, the ultimate aim is to find radiation-sensitive phases in the cell cycle, especially if they are intimately associated with cell division (1-8). A variety of approaches have been devised to single out the response of those individual cells that were irradiated at a given stage in the cell cycle. Cells have been labeled with radioactive precursors of nucleic acids, irradiated, and followed for several generations (1-3). Puck and Steffen (9) have developed a system of mathematical analysis based on the kinetics of cellular proliferation that follows the introduction of a metabolic or radiological block of cell division. This approach permits the localization of radiation lesions in the cell cycle of cells whose pattern of division fits the mathematically derived formulations. Terasima and Tolmach (10) have developed a technique for isolating mitotic HeLa cells from exponentially growing cultures. These cells then divide synchronously for a limited number of generations. They can then be used to study radiation sensitivity throughout the cell cycle. Such a method, essentially representing a technique for synchronization of cell division, has been subsequently modified so that large populations of HeLa cells can be isolated and employed in biochemical studies (11). The technique has also been extended to other cell types (12, 13). Other methods of synchronization of mammalian cells have included the use of nucleic acid precursors or analogs (14, 15) and tem