Dr. S. Rose
The analysis of biological activityre- quires the study of biological organization.
Organization implies some sort of relation between the components making up a com- plex entity. Ideally one should be able to describe every relationship which exists between any one component and all others which make up the complex whole. The very nature of biological organization forces us to use a more practical and sim- plified approach. Thus we study the imme- diate and direct relationship between simple components as they exist to form small complex units, and in the next step we study the relationship between complex units et cetera. This stratifies biological methods into various levels of experimentation. A level of experimentation may be defined by the organizational complexity and number of units which are involved in the study. In these terms an in vitro system which only involves a purified enzyme, inhibitors, acti- vators, substrates, et cetera, is at a lower level of experimentation than a study of the relationship between organs and tissues of the body. Lower levels of experimentation are generally able to examine only simple phenomena. They do so with great pre- cision but often the data does not help to understand how the cell functions. Higher levels of experimentation allow one to study a phenomena as it occurs in the whole organism, but the very complexity of the system reduces the precision of the data and the certainty with which one can interpret the results. Stratification of biological methods has caused a stratification of bio- logical investigators and an increasing gulf divides those who study the animal and its organs as a functional entity and those who
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e attempting to investigate the basic units r°111 which organs and animals are made.This paper will describe especially those techniques which have been designed as
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intermediate experimental levels between established techniques or which have com- bined together a number of experimental levels so that the advantages of higher and lower experimental levels may be utilised.
Intracellular Organization
Cells are not simply bags of enzymes and chemicals mixed in a homogeneous medium freely in solution and subject to classical kinetics. Rather they are a complex hetero geneous system made up of subcellular particles with a special architectural arrangement. The past fifteen years have seen tremendous advances in studying intra- cellular organization which has shed light on to such questions as (1) why are intra- cellular structural units necessary at all, and (2) what effect does structure have on biochemical conditions and reactions in the cell. Certain consistent generalities can be observed, as a result of new data on intra- cellular structures. Thus structure seems to be involved in these situations where the cell requires protection from disrupting agents which the cell must nonetheless include as part of its overall organization.
Enzymes, which if allowed loose in the cell would cause autolysis, can be included in this category. Structure also seems neces- sary in the general situation where synthesis requiring sequental steps and an ordered supply of energy is necessary.
In order to understand the role of intra- cellular organization one must have clear data of the biochemical and structural anatomy of the cellular organization. Tech- niques to obtain this data have various limitations and possible artifacts.
Disruption of cells or homogenization, particularly in aqueous media like sucrose, might yield misleading particles. After all
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one should keep in mind that intracellular injections of aqueous solutions are toxic, and during microsurgical transfer of nuclei from one cell to another care must be taken to avoid the nucleus, coming into contact with the aqueous medium, otherwise it will not survive. These procedures are very much more gentle than gross disruption of the entire cell in an aqueous medium.
Surely under these experimental conditions the possibility exists that a chemical may leave a particle and be found in the soluble cell sap. Equally, it is possible that a chemical leaves one particulate component and becomes attached to another. Despite these obvious pitfalls a vast amount of bio- chemistry is devoted to the study of sub- cellular particles obtained by homogeniza- tion and centrifugation.
Organs or tissues from a multicellular organism are not made up of identical cells.
Consequently homogenizing an organ means that you will be examining the subcellular components of a heterogeneous group of cells. This process, called somewhat face- tiously "Hamburger Biochemistry", imposes another severe limitation on the methods of studying subcellular organization.
Other methods of studying subcellular particles may also be criticised. The elec- tron microscope has become a powerful tool in biology but our dependance on osmium fixation with the possibility of artifacts creates some doubt that too much meaning can yet be attached to detailed appearance and precise measurements of membranes in electron micrographs. Perhaps one of the most important contributions of the electron microscope to biochemistry was the demon- stration that mitochondria of the cell sur- vive homogenization and centrifugation extremely well, and further, one can detect if the mitochondrial prepartion is contami- nated by microsome fragments such as ribonucleoprotein granules or endoplasmic reticulum.
One of the most direct approaches to in vivo biochemical events within subcellular particulates is that of radioautography. The resolution of this method has increased con- siderably by the use of radiohydrogen (tritium) as a label because most of the beta particles emitted by tritium are stopped by the first micron of a photographic emulsion and therefore the grains produced are very
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close to the object being exposed. Other improvements have come through advances in biochemistry which allow the selection of precursors which label a limited number of components of a cell and which allow for differential enzymatic and non enzymatic extraction of specific components of the tissue.
One of the most successful applications of radioautography has been the use of tritiated thymidine as a specific precursor of DNA. The resolution is sufficiently accurate to show labelled and unlabelled segments of single chromosomes. With this technique evidence has been obtained for a duplex type of template for DNA synthesis. Cells or tissues are incubated in the presence of tritiated thymidine for a certain period and then the tritiated thymi- dine is removed. All the chromosomes of a cell appear labelled at the first division following removal of the labelled precursor.
However, these chromosomes reveal their hybrid nature with regard to labelled and unlabelled DNA, for when they duplicate once more in a medium free of labelled precursor, each regularly produces one labelled and one unlabelled daughter chromosome. A radioautographic con- firmation of Mendel's Theory!
The site of synthesis of RNA and its movement in the cell has been studied by a number of techniques. I want to briefly describe one particular set of studies be- cause it combines radioautography and microsurgery. Acanthamoeba were cut with a microneedle controlled by a micromanipu- lator. Following the cutting procedure the nucleated and non-nucleated portions were separated and incubated with labelled RNA precursors. Only the nucleated portions were able to synthesise RNA. Moreover, if a RNA labelled nucleus is transferred by microsurgey to an unlabelled anucleate amoeba the label is transfered across the nuclear membrane to the cytoplasm. These results support the notion that RNA is the carrier of genetic information from the nucleus to the cytoplasm.
I now wish to discuss a particular method of investigating intracellular organization because it demonstrates the value of an intermediate level of experimentation and a combination of techniques. In an earlier section I discussed the almost routine use of
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homogenization and centrifugation to obtain and study the biochemical properties of various intracellular organelles. Despite all precautions any such methods are extremely brutal and one must doubt the identity and properties of the particles so obtained in relation to how they may exist in their natural state.
In order to overcome these difficulties and limitations techniques have been de- veloped whereby cells are centrifuged, their intracellular organelles and components are stratified and can be studied by histo- chemical methods. The fungal hypa offer particular advantages for the study of centrifuged cells. (a) Hyphae are 200 micron long tubular cells enclosed in a resistant wall so that they can serve as ultramicrocentrifuge tubes. The cell dia- meter is about 10 microns and thus strati- fication in a long thin tube can be studied.
(b) The main weakness of most cytochemi- cal reactions lies in their uncertain resolu- tion. However, since hyphae can be con- sidered long, thin ultra centrifuge tubes, separation of the intracellular components overcomes the difficulty of cytochemical resolution. (c) The main advantage of using centrifuged cells in a study of cellular structure lies in the fact that they remain alive and therefore artifacts due to grinding, absorption of chemicals on to a component or loss of a chemical from a component, does not occur. In summary, one is centri- fuging intracellular organelles in an intra- cellular millieu and with a force that does not destroy cell function even though it temporarily and radically stratifies intra- cellular organelles.
This paper will not analyse completely the results obtained with centrifuged cells but will simply describe two main differ- ences obtained with this technique as com- pared to the more regular method of homogenization and centrifugation. In homogenised preparations RNA (soluble) and B galactosidase is found in the super- natant. In the centrifuged hyphae RNA is found in the nucleus, the microsomes and traces in the mitochondria, but none is found in the supernatant. Similarly, in centrifuged hyphae B galactosidase is found bound to mitochondria and not in the supernatant. These examples show that biochemists should not assume with too much assurance that their isolated intra-
cellular organelles are exactly the same as found in the living cell. These examples also demonstrate the value of a combina- tion of techniques and an intermediate level of experimentation namely in vivo centrifu- gation.
Before I leave the exciting area of intra- cellular organization I would like to men- tion some of the micro-surgical and micro- manipulative methods which can be used as tools in this field. With microsurgery one can remove the nucleus of a cell, one can replace a nucleus in an anucleate cell and one can inject various portions of cytoplasm from one cell to another. The Cartesian diver technique can measure the oxygen consumption of single cells. A torsion bal- ance has been devised which has a repro- ducibility of 0.01 of a microgram. Using interference microscopy the protein content of a single cell can be determined, and by X-ray absorption the density of cellular and subcellular particles can be measured. An ingenious adaptation of chromatography has been described which permits determination of the total quantity of RNA from a single cell. This RNA can be hydrolysed and the nucleotite anaylsis of the RNA of a single cell can be obtained by chromatography on a copper silk fibre. This method is one million times more sensitive than conven- tional methods. The above is by no means a complete list of all the microsurgical, micromanipulative and microanalytical methods which are available.
The question naturally arises as to why such sensitive methods are required. After all, we are often able to collect large samples of biological material. The answer lies in the heterogenicity of biological materials. The inside of a cell is not homo- geneous and a tissue is not composed of a homogeneous collection of cells. In order to study one portion of a cell, or in order to study one cell, micromethods must be used.
If we make estimations of the whole mass, we are making estimations of mixed popu- lations of biological particles and averaging out the result.
Interorgan Relations
The functional activity of many biologi- cal systems is regulated by more than one mechanism. Some of these mechanisms are in the form of servo controlled systems and
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others are specifically designed to alter the activity level of the system. Each mechan- ism is composed of a number of sequential steps involving different anatomical and functional units. These mechanisms are inter-related either at the final active tissue cell or at one of the intermediate steps of the several mechanisms.
The analysis of such controlled systems involves an anatomical and physiological mapping of the several steps of each mech- anism and the inter-relationship between them. Such studies are a pre-requisite to the more detailed investigations of each step in terms of biochemical mechanisms.
The main difficulty in the design and interpretation of such experiments is de- termining the primary site of action of administered compounds. This difficulty has been overcome when the compound acts quickly and when operation, anaesthesia and exposure of the area do not interfere with the experiment. Thus acetylcholine has been shown to be involved in the trans- mission of impulses at the neuro-muscular junction.
But let us suppose that acetylcholine was
Figure I.
acting on one particular tissue to effect a or duration. The usual methods are no longer applicable. If the acetylcholine is given intravenously it will not reach the biological phenomena of slow development target site in sufficient concentration to be effective. If it is given intra-arterially as a single injection the (slow developing) bio- logical response will not occur. These diffi- culties have been overcome by the use of a continuous injector which can be buried subcutaneously or attached to the animal.
The injector can deliver drugs continu- ously and constantly for many weeks to an unanaesthetised unrestrained animal. More- over, the drug is delivered directly to the site of interest by a fine polythene tube (50- 200 microm diameter) which is buried in the animal. There are many obvious advantages of this technique. Thus (a) for compounds whose activity is quickly de- stroyed in vivo local infusion allows a physiological or pharmacological level to be achieved at the site of interest. (b) The local dose of drug is so arranged as to pro- duce a significant concentration at that site without an effective systemic concentration being reached. The local effects obtained may then be considered to be a direct effect on that tissue rather than a sequel to a change elsewhere in the animal. (c) The chemical structure of the drug at the extra- cellular fluid level of that tissue is known more precisely. (d) Many biological phe- nomena of slow development may be studied—thus the technique can be used to study the effect of prolonged altered chemical environment on the secretory function, metabolic activity, morphogenesis and growth of a variety of tissues. (e) Con- tinuous administration approaches more closely to normal physiological conditions when dealing with replacement type studies.
To gain the above advantages workers have resorted to in vitro tissue culture tech- niques. This present approach has been envisaged as a bridge in the gap between
in vitro tissue culture and whole animal studies so that certain disadvantages of both levels of experimentation can be avoided if necessary.
A brief description of the technical aspects may be of interest. The motive power for the injector is the osmotic pres- sure developed by a saturated solution of
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