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This paper describes information-handling aspects of the TYCHO I analysis system (Clin, Chem. 27: 1807--1820, 1981), which analyzes two-dimensional electrophoresis gels, matches the individual protein spots with those in a reference pattern, and stores various information--including spot measurements, identifications, treatment profiles, set memberships, and comments--in a computerized database. This and additional information such as amino acid composition and cellular localization is then accessible from an interactive program that includes a pictorial user interface and presents much of the data in graphical form. Use of the TYCHO I system is illustrated by examples drawn from analyses of gel patterns from human leukocytes.

We have been developing a clinically useful method for high-resolution two-dimensional electrophoretic analysis of small (5--10 mg) human muscle biopsy samples with sufficient resolution to resolve the major contractile proteins and enzymes. Using rabbit psoas muscle as a model, we describe methods for sample preparation and two-dimensional electrophoresis. Basic proteins, which appear as streaks when conventional isoelectric focusing is used in the first dimension, are resolved through a modification of the nonequilibrium pH gradient electrophoresis method [Cell 12, 1133 (1977)]. In the two-dimensional patterns obtained from rabbit muscle, we identify the components of 10 enzymes and of myosin, actin, tropomyosin, and troponin. These patterns indicate charge heterogeneity in a large fraction of the proteins. Comparison of rabbit and normal human muscle patterns shows many similarities, but much additional work is required to confirm identifications. We conclude that analysis of small biopsy samples is feasible, but that all aspects of human sample acquisition, storage (when necessary), and preparation require thorough study before the method becomes routine in human muscle research and, ultimately, in the diagnosis of some muscle diseases.

The ISO-DALT two-dimensional electrophoretic system (1,2), based on the method of O'Farrell (3), is capable of performing large numbers of analysis on complex mixtures of proteins. However, both separations employed are carried out under dissociating or denaturing conditions and no enzyme activities are readily observable in the analyzed proteins. In order to identify the spots corresponding to particular enzymes, it is therefore necessary to employ some nondestructive resolving technique first and as a second step to perform both enzyme and two-dimensional electrophoretic analyses on the fractions generated. By correlating enzyme activity with intensity of various spots on the two-dimensional gels throughout the series of initial fractions, identifications, can be made. This approach, unlike the more direct immunoprecipitation methods (4), requires the running of large numbers of enzyme analyses and two-dimensional gels and some convenient initial resolving procedure. Convenient and rapid techniques for the analyses (5,6) and gels (1,2) have been described previously in this series and elsewhere. This paper deals with the use of selective denaturation in a temperature gradient as an initial resolving procedure and describes a simple thermal gradient device for generating such a gradient.

Human erythrocyte lysate proteins were resolved into over 250 discrete spots by two-dimensional electrophoresis using isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate, (SDS) in the second. The overwhelming excess of hemoglobin has made such analyses difficult in the past. However, with the ISO-DALT two-dimensional electrophoresis system, large numbers of red cell proteins can be mapped in the presence of hemoglobin. When hemoglobin and several other major proteins are removed by adsorption to DEAE-cellulose, additional minor components are seen, giving a total of over 275. With the use of purified preparations, the map positions of five cell enzymes or their subunits were determined: pyruvate kinase, catalase, glucose-6-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, and carbonic anhydrase. The mapping techniques described complement and extend those traditionally used to find human red cell protein variants.

Principles and applications of an automatic cyclic affinity chromatography system, especially as applied to separation of proteins by immunosorption, are described. Use is made of columns of either immobilized antigens or antibodies to separate from a mixture, in a repetitive fashion, a desired protein or proteins by immunoadsorption followed by elution or to take from a mixture all proteins but the desired ones by allowing these latter to go through unadsorbed. The amplification provided by the cyclic use of the system and the biological amplification of hyperimmunization in achieving useful yields of desired proteins is discussed. Experience in the use of eluting solutions which do not cause damage to either the fixed or eluted proteins is presented. Representative separations done with the system are described, illustrating the different modes of use.

Procedures to measure two important properties of two-dimensional electrophoresis systems have been developed. These procedures evaluate the overall resolution and positional reproducibility of two-dimensional gel patterns. Using these measures we show that various state of the art gel systems can produce patterns exhibiting resolution measures of 15 000–50 000 (yielding practical upper bounds of about 2000–6000 detectable proteins in typical cellular samples) and that positional accuracy for most spots in gel-to-gel comparisons is better than a spot width (0.5–2.0 mm). Implemented as part of a computerized image analysis system for two-dimensional gel data, these two measures can be used in the selection and optimization of gel systems, and in the selection of sets of well-resolved, well-matched spots for genetic and other quantitative sudies.

Design features and operation of a prototype miniaturized Fast Analyzer are described, and some results obtained with it are presented. The Analyzer occupies only one cubic foot of space. It has a 17-cuvet plastic rotor that rotates through a stationary optical system at speeds up to 5000 rpm. The resulting centrifugal force is utilized to transfer and mix a series of sample(s) and reagent(s) into the cuvets. The ensuing reactions are monitored spectrophotometrically, and the data evaluated in real time by an on-line computer. Samples (1 to 10 µl) and reagents (70 to 110 µl) are loaded into the rotor either discretely or dynamically; various rotor configurations can be used to do this. Many of the standard clinical analyses, including most of the NADH-linked enzymatic analyses, have been adapted for use with this analyzer. Precision obtained ranges from 1 to 4%. This report considers, specifically, analyses of some serum enzymes. Results show that the small analyzer possesses the previously demonstrated advantages of Fast Analyzers and, in addition, has several beneficial features arising from miniaturization.

1. 1. Treatment of dilute rat thymus breis with concentrated NaCl solutions produced viscous solutions that were permanently disrupted by shearing. Attempts to measure the viscosity of such extracts in ordinary capillary viscometers were unsuccessful; however, reproducible measurements of apparent relative viscosity (± 2–3 per cent) were obtained by timing the first passage of the extracts through a modified Cannon-Fenske viscometer. 2. 2. The viscosity of extracts prepared in 1 M NaCl reached a maximum and constant value only after 10–12 hours of extraction at room temperature (25–27 °C). 3. 3. The maximum apparent relative viscosity of the extracts varied logarithmically with homogenate concentration. 4. 4. The viscosity of the extracts was markedly affected by pH. From pH 6.4 to pH 8.5, the viscosity was relatively constant; below pH 6.4, it fell rapidly, and, at lower pH's, was the same as for the solvent. Above pH 8.5, the specific viscosity increased rapidly to a maximum of about pH 10 and then dropped to zero at pH 11.5. 5. 5. The viscosity of the extracts was low in 0.05 to 0.4 M NaCl concentrations but increased rapidly in concentrations of 0.6 to 0.9 M. The viscosity of the extracts continued to rise slowly as the NaCl concentration was raised to 2 M but then decreased when the concentration was raised above 2 M. 6. 6. Viscosity decreased when the extracts or the breis from which the extracts were prepared were frozen. 7. 7. The results indicate that at least two types of protein-DNA bonds exist. The first, which cross-links DNA-protein strands in the nuclear gelwork, is easily broken by 1 M NaCl. The second, which binds the DNA molecules end to end to form long strands, is not broken in the initial extract and accounts for the high viscosity. The strands are believed to be the fundamental units of chromosome structure.

Sample analysis rate for a GeMSAEC Fast Analyzer is directly proportional to the number of sample cuvets contained in each rotor. GeMSAEC Fast Analyzers are available having either 15-, 16-, or 30-place rotors. To increase the sample analysis rate, an advanced GeMSAEC Fast Analyzer has been designed and fabricated that uses a 42-place rotor. The 42-place rotor, in order to accommodate the increased number of cuvets, is a modified enlargement of the previously described 15-place rotor [N. G. Anderson, Science 166, 317(1969)]. The hardware of the 42-place system is fully compatible with the existing GeMSAEC computer module, and existing software used in the 15-place system has been modified for use with the 42-place system. Results obtained for various chemical and enzymatic analyses are discussed.

The distribution of catalase, rhodanese, acid phosphatase, nucleoside phosphorylase, esterase, adenosine deaminase, glutathione reductase, and β-glucuronidase in the free-electrophoresis pattern of the soluble phase of rat liver at pH 7.5 has been determined.

The taper in the K-II core was designed to aid in maintaining resolution during dynamic unloading. Static unloading has proved to be superior in practice however, thereby eliminating the requirement for a taper. Since, as shown here, superior recovery is obtained with a nontapered core, the latter design has been adopted for the K-III titanium continuous-sample-flow-with-banding rotor (17).

A number of artefacts which prevent ideal sedimentation have been found during the centrifugation of rat liver breis. These include hydrodynamic effects which cause local areas containing a number of large particles to move as a unit dragging along associated smaller particles, turnover effects at density boundaries, convection currents due to heat and mechanical agitation, and wall effects. Both breis and suspensions of red cells have been used in these studies. A separation approaching the ideal is obtained by the use of more dilute breis, careful layering over a continuous density gradient sufficiently steep to prevent the anomalies mentioned, and a rigid adherence to a gradual acceleration and deceleration schedule. Gradients of known characteristics are produced by a mechanical gradient engine. A stroboscopic illumination system has been devised for studying the behavior of layered systems in a refrigerated centrifuge having an observation port. A speed-versus-time recording system allows integration of the centrifuging done. Speeds are accurately measured to 0.02 per cent. New sector-shaped centrifuge tubes which minimize wall effects and a sliding device for recovering all levels of the gradient from the tube after centrifugation are described. Breis may now be quantitatively fractionated in one or two steps.

1. 1. One molar NaCl extracts of rat thymus are highly sensitive to X rays, and an exposure of 10 r causes a detectable loss in viscosity. An aftereffect was observed, and the viscosity of irradiated samples continued to decrease for 4–5 hours after irradiation. Dose curves were sigmoid. The effects of X rays on the extracts were largely indirect. Freezing, the addition of chemical protective agents, and increasing the concentration of the gell all had a dosereducing effect. There seemed to be a small contribution from direct effects not exceeding a few per cent of the total. 2. 2. Except for cyanide, all of the compounds tested that are known to protect animals from the lethal effects of irradiation also protected in the extracts. However, several compounds that had no protective action on animals gave large dose-reduction factors in the extracts. No good correlation exists between chemical agents protecting animals and those giving dosereducing effects in the extracts. 3. 3. The high sensitivity of the extracts to X-irradiation appeared to be a direct consequence of the high dilutions employed. Direct irradiation of the homogenate and excised tissue required, respectively, 100 and 700 times the X-ray dose necessary to produce the same effect in the extracts. The higher doses probably reflect protection from indirect effects by the high concentration of DNP in the cell nucleus and by protective substances such as proteins. The fact that one can demonstrate an effect by direct irradiation of the tissue is of interest, since no effects on DNA have been detected after irradiation in vivo .

Basic research necessary for the development of advanced methods for clinical chemistry are reviewed. It is concluded that a new class of fast analyzers are required which are inherently adapted to computer data reduction. A new analytical system using centrifugal force to transfer and mix fluids in a multiple-cuvet rotor yields data signals at intervals of 3.3 milliseconds. It is applicable to a variety of analyses, but is especially suited to the determination of enzymes by measuring reaction rates.

A cuvet-rotor, designated G-IIB, has been developed that contains small-bore syphons attached to each of the fifteen cuvets. Drainage of reactants from the transfer disc was found to be approximately 90% complete in 1 second and 99% complete after 2.5 seconds during acceleration to 1000 rpm. Mixing of protein-biuret reagent solution and 30% sucrose occurred in 1 second when a vacuum was used to pull air back through the syphons and cuvets. Drainage of the cuvets through the syphons was almost quantitative (>99%) when air pressure and one cycle from 400 rpm to 1000 rpm and back to 400 rpm was used. The interval between successive sets of analyses may therefore be 1 minute or less. A method of heat cycling to make the Telflon cuvet-spacer conform to the glass windows is presented.

SummaryThe specific induction of transplantation immunity against adenovirus 31-and SV40-induced hamster tumor cell challenge employing either a crude membrane fraction of homologous adenovirus 31 tumor cells or SV40 tumor cell fragments is described. In each case immunity was equivalent or superior to that induced by whole, irradiated tumor cells previously described to contain TSTA, or to that induced by vaccination of mature hamsters with the live virus. Whole tumor homogenate of adenovirus 31 hamster cells, from which the membrane fraction was obtained failed to induce TSTA immunity. The possibility exists that a masking or enhancing component is present in the homogenate to interfere with TSTA immunity induction or detection.Addendum. Hamsters immunized with SV40 tumor cell ghost lysates were uniformly resistant to an initial cell challenge as shown in Table III. Several months later the survivors were challenged a second time and 42% of the hamsters developed tumors by day 50. One hundred percent of...

SummaryDifferential and zonal ultracentrifugation followed by brief ether exposure or by isopycnic banding in CsCl, has yielded virus-like particles from the plasma of 8 leukemic patients. In electron microscopic examinations, two morphologically different particles were observed, designated “R” and “Q.” In 18 controls, no “R” particles were found, but in one control “Q”-like particles were observed. The density of the particles is the same in all instances.

Information on the condensation of chromatin by organic substances is of interest in relation to attempts to formulate the properties of the chromosomal condensant active during cell division. The relation of nuclear volume to concentration has therefore been studied. n -Propylamine, an example of an aliphatic monoamine, produced swelling in low concentrations (0.001 to 0.01 M ) but little volume change in the range 0.025 to 0.36 M , closely paralleling results obtained with NaCl and KC1. Diamines (diaminopropane, -butane, -pentane, -hexane, -heptane, and -decane) produced shrinkage and granulation and nucleolar condensation in ≈ 0.025 M solutions and less shrinkage as the concentration of the diamines was increased. Condensation was readily reversed by either distilled water or the isolation medium. The effects of diamines resemble those of MgCl 2 and CaCl 2 . Spermidine and spermine, which possess three and four positive charges, respectively, produce condensation at very low (0.001 M ) concentrations, with marked swelling at approximately 0.2 M . Condensation was not reversed by distilled water and was slowly reversed by the phosphate-containing isolation medium. Chromosome-like structures were observed in a number of solutions but accurate counts were not possible. In a comparison of Amphiuma erythrocyte nuclei and rat liver nuclei, relatively few granules were seen in the former (2n = 24) whereas many were seen in liver nuclei (4n = 84), suggesting that the structures observed may be chromosomal in origin. Available evidence suggests that the prophase condensing agent contains more than two charges if condensation is caused only by charge effects.

Over 300 compounds have been reported in human urine (1-4). These include inorganic compounds, organic acids, sugars, amino acids, purines and related compounds, enzymes, hormones, vitamins and their metabolites, estrogens, and many other organic compounds. The quantities of these constituents found in individual urine samples represent a wealth of information that can be used to evaluate body functions. Such quantitative data have been collected on a limited basis for many years and, as a result, many molecular constituents have been shown to have pathological significance. For example, a high uric acid content is associated with gout(5) and some leukemias (6); a high urocanic acid content is sometimes found in conjunction with asthma(7); large amounts of phenolic and indole amines appear to be excreted by schizophrenics(8), etc.The Molecular Anatomy (MAN) program at Oak Ridge National Laboratory(9) is concerned with the description, at the molecular level, of the structure and organization of cells and ...

New instrumentation has been developed at ORNL for analyzing physiologic specimens. The system is designed for use in clinical laboratories, hospitals, and emergency rooms and for use by researchers. Miniaturized versions may also be used in prolonged space flights. After consideration of ease of automation, flexibility, and rapid startup, multiple analyses performed in parallel was chosen as the desired approach. Concepts employed in developing the system are discussed, including: (1) dynamic mixing of samples and reagents through centrifugal transfer from transfer disks into cuvets; (2) rotor design allowing emptying, flushing, and drying of cuvets; (3) precise temperature control; (4) spectrophotometric reaction monitoring; (5) data reduction, with all data stored for possible recall; (6) feedback control for error correction and time saving. Possible future improvements are indicated.

Ambrose et al. propose a likely scheme for the course of tumour development, as a failure of the host immune system.

Summary Performance of the K-II continuous-sample-flow-with-banding zonalcentrifuge rotor was evaluated using sucrose gradients and polystyrene latex beads having a mean diameter of 0.091 μ and a banding density of 1.049 gm/cm3 in sucrose at 20°C. Narrower particle zones were recovered with static unloading as compared with dynamic unloading. With the former, the average band width at half-height was 64 ml in the total volume of 3600 cm3, corresponding, to a width of 0.24 mm in the rotor. Multiple reorientations of the gradient produced only a small loss of resolution. Measurements of gradient decay and sucrose loss during continuous flow indicate that gradients suitable for influenza virus recovery may be retained in the rotor for 10 hr, while 200 liters of fluid passes through the rotor. The particle capture efficiency of the rotor is slightly less than predicted by the Berman theory.

A simple gradient device is described which allows convex gradients to be formed for use in zonal centrifuge rotors. The configuration chosen produces very rapid mixing of the very viscous sucrose solutions. By varying the total volume of the mixer, the shape of the gradient may be altered within rather wide limits. With large volume mixers, gradients may be obtained which are very nearly linear with radius when placed in the sector-shaped compartments of the rotor. With smaller volume mixers, gradients that are convex with respect to radius when in position in the rotor may be generated which are more useful where high-capacity high-resolution separations are required.

This discussion has included only a partial list of the systems now under development at Oak Ridge as part of the feasibility studies for the Molecular Anatomy Program. It is evident that we are still in the "Robert Goddard" phase of this work. It may not be premature, however, to suggest several conclusions. Biomedical scientists are discouraged on discovering that developmental efforts cost more, by one or two orders of magnitude, than pure research. In part this is because the full cost of development is generally shown, while in pure research some of the costs may be hidden, or the funds supplied by several sources. Regardless of the reason, the fact remains that development is expensive, as is well understood in nuclear physics and space science. The role and mission of the large national laboratories, and the kinds of research that should be done in them, have been discussed by Weinberg (63). The studies described here were in part stimulated by his ideas. We have been unable to find an environment outside a large national laboratory where a program like the Molecular Anatomy Program could be undertaken at the present rate. It appears that programs which attempt to make use of the multidisciplinary approach characteristic of national laboratories should be carefully designed and should evolve experimentally. There is less chance of success when a program is an administrative invention than when it evolves from scientific invention and discovery. It has been pointed out (64) that most program decisions in science are secret decisions in the sense that the scientific community as a whole does not participate in them. If a choice is to be made at some future time between large-scale expenditures for exploring space, for developing new weapons systems, for constructing new accelerators, for designing large reactors, or for systematically developing methods to explore the molecular basis of human disease, then we will need sufficient information to evaluate each alternative fully, and the information should be generally available. It appears desirable, therefore, to allow the Molecular Anatomy Program to proceed to a point where the full range of its contributions and its inherent limitations may be seen. A rational choice may then be made.

An optical density thermometer is described for determining the temperature of centrifuge rotors. Quantitation is based on the change in the concentration of the vapor phase of a volatile substance, such as bromine, with temperature. Centrifugal force holds the liquid phase away from the light path and removes condensation from the optical windows.

SummaryNonvirion T antigen was isolated from adenovirus-type-31 solid tumors by a series of differential extractions, including overnight digestion with papain, centrifugation, acetone and ammonium sulfate precipitation, and acrylamide-gel electrophoresis on a vertical flat-bed apparatus which enabled us to isolate individual bands of protein. The antigen is membrane-associated, has a sedimentation coefficient of 2.93, is partially heat labile, stable to freeze-thaw, and travels rapidly in acrylamide gel. It is probably not the only T antigen moiety present in these tumors.

The plasmas of 255 patients with neoplasms of the blood-forming organs and 44 controls were screened for "virus-like" particles. Plasma samples were banded by cesium chloride density-gradient centrifugation and negatively stained, and representative electron-microscope fields were photographed and particles counted. The number and distribution of particles were evaluated according to diagnosis, clinical status of the patient, white-cell count and platelet count. No relation between any of the classes of partides and clinical features were found. A large percentage of both control and disease groups had about the same numbers of particles. No common clinical characteristics of patients with extremely high particle counts were found. "Virus-like" particles seen by this technic in the plasmas of patients with leukemia and related disorders are considered to be unrelated to their disease. The importance of thin sectioning in future studies of this kind is emphasized.

1. Two months after injection of rat bone marrow into lethally X-irradiated mice (950 r-RBM mice), 100% of the circulating RBC were serologically of the rat type, indicating that the surface molecular configuration of RBC from these experimental mice are of the rat type. 2. The hemoglobin was found to be also very much like the rat type in its ease in crystallization, its alkali denaturation property, its electrophoretic property, and its tendency to form a paracrystalline state at low temperature. 3. These cells possessed dual osmotic properties; the relative hemoglobin concentration released when cells were lysed in water was more comparable to the rat type, but its temperature dependency was more comparable to the mouse type.

The purpose of this review is to summarize the present status of preparative zonal centrifugation and to indicate where the remaining problems, in the reviewer's opinion, may lie. The field is sufficiently new for interest in practical separartions to be uppermost. However, a number of theoretical problems relating to gradient capacity, zonal rotor configuration and particle—particle interactions remain to be fully explored.

The zonal ultracentrifuge was used for separation of Treponema pallidum from large volumes of rabbit testicular syphiloma extracts by continuous-flow centrifugation in a cesium chloride density gradient. The gradient was linear with radius from a density of 1.05 to 1.36 g/ml. Operating speeds were 15,000 rev/min for the continuous-flow phase and 25,000 rev/min for a 30-min banding period. A total of 9 × 10 9 (24.3%) treponemes were recovered from the original extract. Of the treponemes recovered, 88% formed a band at a density of 1.170 to 1.211 g/ml. Within the limits of present methods of assay, these fractions were relatively free from testicular particulates and protein when compared with treponemes recovered after differential centrifugation. Observations of the isolated fractions by dark-field and electron microscopy indicated a lack of gross morphological damage to T. pallidum . Their antigenic characteristics were also retained, as evidenced by their ability to react with syphilitic sera in the indirect fluorescent-antibody procedure.

An inexpensive yet versatile device for driving the mechanical stage of a microscope is described. The device provides for continuous variable-speed control and positively monitors the distance traversed at short and known increments of stage travel. This device was attached to the horizontal stage of a microscope used as the optical component of a microdensitometer in order to facilitate the accurate and precise determination of migration distances when scanning developed electrophoresis patterns.

A simple system, based on a series of vessels arranged so that the top of each is at the midlevel of its predecessor, and all draining by gravity into a common line, has been shown to be capable of producing very complex, predictable gradients. By simply changing the levels, so that the top of each chamber is below the level of its predecessor, a step elution system may be obtained.

The gel released from isolated rat liver nuclei in response to heparin treatment has been found to stain with methylene blue, azure A, and methyl green when the dyes were added to the salt-sucrose nuclear isolation medium. Azure A and methylene blue caused rapid nuclear shrinkage to as little as 1 4 the original nuclear volume. Subsequent treatment with heparin caused the nuclei to fade rapidly and swell to approximately 5 4 of the original volume. With methylene blue stained nuclei heparin caused the extrusion of deeply stained, slightly birefringent rods through apertures on the nuclear surface. Methyl green also caused nuclear shrinkage, but to a lesser degree. Studies with the Feulgen reaction demonstrated structural damage in isolated rat liver nuclei as a result of heparin action. The viscous material released by heparin was shown to be Feulgen positive by resort to hydrolysis without prior fixation, since after customary fixatives the presence of a Feulgen positive reaction outside the nucleus could not be clearly demonstrated. The possibility is suggested that the Feulgen reaction following the customary fixatives depends in part on the manner in which the DNA is bound. The nuclei of leucocytes with visually intact cell membranes included in the nuclear preparations failed to show structural damage due to heparin and it is suggested that either the cell membrane provides some protection against heparin action or that damaged cells are more susceptible to this action. Observations made provide additional basis for the conclusion that heparin replaces DNA in the nucleo-histone of the nucleus, resulting in the structural damage observed, and releasing DNA in the form of a soluble viscous protein containing complex.

1. The effects of morganic ions, electrolyte concentration, and pH on the appearance and volume of the isolated rat liver nucleus have been studied. Nuclei were isolated by differential centrifugation in a buffered salt-sucrose mixture at pH 7.1. Nuclear volumes were determined photographically. 2. In solutions of NaCl, of KCl, and in potassium phosphate buffers the nuclear volume decreased markedly with an increase in concentration from 0.001 M to 0.05 M but remained essentially constant with further increase in concentration to 1.0 M. The effects of CaCl2 and MgCl2 differed from those of NaCl and KCl in that a smaller volume was obtained in concentrations less than 0.15 M, and in the case of CaCl2 an increase in volume was obtained in more concentrated solutions. The volume changes are considered to be due primarily to ionic effects on the nuclear colloids rather than to osmotic behavior. 3. Treatment of nuclei with DNAase prevented the characteristic volume changes resulting from ion effects, suggesting the importance of DNA in nuclear volume changes. 4. The optical changes in isolated nuclei in various concentrations of KCl, NaCl, CaCl2, MgCl2, and in potassium phosphate buffers as observed under phase contrast illumination are described. CaCl2 gave the most marked nuclear changes from the conditions in the uninjured cell and caused shrinkage and granulation in 0.001 M concentration. The effects of CaCl2 were also manifested in 0.88 M sucrose, in mixtures with monovalent salts, and in serum. Changes in nuclear volume and optical appearance which occurred in salt solutions and in 0.1 N HCl were readily reversible. 5. Nuclear volume remained constant between pH 8.91 and 5.12 and decreased in more acid solutions. 6. Sucrose had no appreciable osmotic effect, and in hyperosmotic solution. (0.88 M) nuclei showed swelling and rupture comparable to that in distilled water. 7. The results are considered in relation to the requirements of nuclear isolation media. 8. Rat liver nuclei isolated in a buffered salt-sucrose medium by differential centrifugation exhibited a pattern of size distribution similar to that of fixed nuclei but were of considerably larger volume. The ratio of the volumes of the peak frequencies of the two chief size groups was 1:1.9.

Supernatants of rat skeletal muscle homogenates were fractionated by differential centrifugation and by zonal centrifugation in sucrose density gradients. Cytochrome oxidase was employed as an enzymatic marker for locating mitochondria. The subcellular fractions were also assayed for their ability to prevent the ATP-induced contraction of myofibrils. Both the mitochondrial and microsomal fractions obtained by differential fractionation were found to be rich in such relaxing activity, and the microsomal fraction was appreciably contaminated by mitochondria. In contrast to this, when fractionation was carried out by means of zonal centrifugation (4200 RPM x 205 min. to 40,000 RPM x 60 min.), relaxing activity was found to be associated only with particles having the sedimentation characteristics of microsomes (s20,w estimated to be between 370 and 1880S). Relaxing activity was not detected in the regions of the gradient containing either the starting sample zone (soluble phase) or the mitochondrial peak. The microsomal relaxing particles showed negligible cytochrome oxidase activity.

The design and fabrication of a miniature Fast Analyzer that utilizes a small, multicuvet, plastic rotor and a unique optical system to measure the light transmittance of each cuvet is described. The system was developed to accept small, discrete volumes of reagent and sample, which are then transferred and mixed in less than 10 seconds. A serum albumin analysis was performed to indicate the feasibility of miniaturization.

The zonal ultracentrifuge was used to separate the subcellular components of rat liver brei into soluble phase, microsomal, mitochondrial, membranous fragments, and nuclear fractions during a single centrifugation. The centrifuge was run at 10,000 to 30,000 RPM for 15 to 240 minutes, and the rotor contained a 1200 ml sucrose gradient, varying linearly with radius from 17 to 55 per cent sucrose with a "cushion" of 66 per cent sucrose at the rotor edge. The distribution of the mitochondria was determined using cytochrome oxidase as the marker enzyme. An automated assay system for cytochrome oxidase was developed utilizing reduced cytochrome c as substrate, modules of the Technicon Autoanalyzer, and the Beckman DB Spectrophotometer. All of the cytochrome oxidase activity was restricted to a single peak in the gradient, and no activity could be detected in the zones occupied by the microsomes and nuclei. The mitochondrial fraction was isolated from rat liver brei in 0.25 M sucrose by differential centrifugation, and then run in the zonal ultracentrifuge.This fraction behaved in the zonal ultracentrifuge in the same way as mitochondria separated directly from intact brei. Observations of the isolated fractions in the phase contrast microscope indicated that a wide variety of granules was present in the mitochondrial zone in addition to the true mitochondria. Under the conditions employed, the mitochondria were sedimented essentially to their isopycnic position in the gradient at approximately 43.8 per cent sucrose, density 1.20 gm/cc.