Stimulation of cell division. Cell division. Need fasting days

The optimal stage for studying chromosomes is the metaphase stage, when the chromosomes reach maximum condensation and are located in one plane, which allows them to be identified with high accuracy. To study a karyotype, several conditions must be met:

Stimulation of cell divisions to obtain the maximum amount dividing cells,

- blocking cell division in metaphase;

- hypotonization of cells and preparation of a chromosome preparation for further examination under a microscope.

To study chromosomes you can use cells from actively proliferating tissues(bone marrow cells, testicular walls, tumors) or cell cultures, which are obtained by culturing under controlled conditions on special nutrient media of cells isolated from the body (peripheral blood cells*, T lymphocytes, red bone marrow cells, fibroblasts of various origins, chorion cells, tumor cells)

* The technique of obtaining chromosomal preparations from peripheral blood lymphocytes cultured in isolated conditions is the simplest method and consists of the following steps:

Venous blood collection under aseptic conditions;

Adding heparin to prevent blood clotting;

Transfer of material into vials with a special nutrient medium;

Stimulation of cell division by adding phytohemagglutinin;

Incubation of the culture for 72 hours at a temperature of 37 0 C.

Blocking cell division at the metaphase stage achieved by introducing into the medium colchicine or colcemid substances - cytostatics that destroy the spindle. Receipt preparations for microscopic analysis includes the following stages:

- hypotonization of cells, which is achieved by adding a hypotonic solution of potassium chloride; this leads to cell swelling, rupture of the nuclear membrane and chromosome dispersion;

- cell fixation to stop cell activity while preserving the chromosome structure; for this, special fixatives are used, for example, a mixture of ethyl alcohol and acetic acid;

- staining of the drug according to Giemsa or the use of other staining methods;

- analysis under a microscope in order to identify numerical disturbances (homogeneous or mosaic) And structural aberrations;

- photographing and cutting out chromosomes;

- identification of chromosomes and compilation of a karyogram (idiogram).

Stages of karyotyping Differential coloring of chromosomes

Currently, along with routine methods of studying the karyotype, differential staining methods are used, which make it possible to identify alternating colored and unstained bands in chromatids. They're called bands and havespecific Andexact distribution due to the peculiarities of the internal organization of the chromosome

Differential staining methods were developed in the early 70s of the twentieth century and became an important milestone in the development of human cytogenetics. They have wide practical applications, because:

The alternation of stripes is not random, but reflects internal structure of chromosomes, for example, the distribution of euchromatic and heterochromatic regions rich in AT or GC DNA sequences, chromatin regions with different concentrations of histones and non-histones;

The distribution of bands is identical for all cells of one organism and all organisms of a given species, which is used for accurate identification of the species;

The method allows you to accurately identify homologous chromosomes, which are identical from a genetic point of view and have a similar distribution of bands;

The method provides accurate identification of each chromosome, because different chromosomes have different distributions of bands;

Differential coloring allows us to identify many structural abnormalities of chromosomes(deletions, inversions), which are difficult to detect using simple staining methods.

Depending on the method of chromosome preprocessing and staining technique, several differential staining methods are distinguished (G, Q, R, T, C). Using them, it is possible to obtain an alternation of colored and uncolored bands - bands, stable and specific for each chromosome.

Characteristics of various methods for differential chromosome staining

Method name

Dye used

The nature of bands

Practical role

Painted -

heterochromatin;

unpainted -

euchromatin

Detection of numerical and structural chromosome abnormalities

Quinacrine (fluorescent dye)

Painted -

heterochromatin;

unpainted -

euchromatin

Method R (reverse)

Colored - euchromatin;

unpainted -

heterochromatin

Detection of numerical and structural chromosome abnormalities

Giemsa or fluorescent dye

Stained centromeric heterochromatin

Chromosome polymorphism analysis

Giemsa or fluorescent dye

colored - telomeric heterochromatin

Chromosome polymorphism analysis

Stimulators of cell metabolism and stimulators of regeneration: placenta extract, amniotic fluid extract, panthenol, extract of medicinal leeches, salmon milk, sea plankton, pollen, bone marrow, embryonic cells, royal jelly of bees (apilak), DNA, RNA, growth factors, organ preparations thymus, umbilical cord, bone marrow, sea buckthorn oil, phyestrogens, etc.

Growth factors are proteins and glycoproteins that have a mitogenic effect (stimulate division) on various cells. Growth factors are named after the cell type for which mitogenic action was first shown, but they have a wider spectrum of action and are not limited to one group of cells. Keratinocyte growth factor stimulates keratinocyte division. Appears when the skin is wounded. Epidermal growth factor - stimulates regeneration. Suppresses differentiation and apoptosis, ensures reepithelialization of wounds. May induce tumor growth. Heparin-binding growth factor has an antiproliferative effect on keratinocytes. Nerve cell growth factor stimulates keratinocyte division. Currently, growth factors capable of activating human cell division have been isolated from whey, animal amniotic fluid, placenta, human embryonic tissue, gonads of invertebrate animals and mammalian sperm. Growth factors are used to activate mitoses in aging skin, accelerate epidermal renewal and skin regeneration.

What substances stimulate cell renewal?

  • Vitamins,
  • microelements,
  • amino acids,
  • enzymes,

These could be: vit. A, E, C, F, zinc, magnesium, selenium, sulfur, silicon, vit. group B, biotin, glutathione, protease, papain, etc.

Substances that increase skin turgor and elasticity, elastic stimulants (sulfur, vitamin C, chondroitin sulfate, hyaluronic acid, collagen, silicon, glucosamines, retinoids and retinoic acid, fibronectin, phytoestrogens, cellular cosmetics, etc.).

Retinoids

Retinoids are natural or synthetic compounds that exhibit an effect similar to retinol (vit. A). The effect of retinoids on the skin: exfoliating, brightening, increasing firmness and elasticity, smoothing wrinkles, reducing inflammation, wound healing, side effect - irritating. Retinoids cause simultaneous thickening of the epidermis and exfoliation of the stratum corneum, accelerating the turnover of keratinocytes. Retinoid groups:

  • Non-aromatic retinoids - retinaldehyde, tretinoin, isotretinoin, trans-retinol b - glucuronide, fentretinide, retinoic acid esters (retinyl acetate, retinyl palmitate).
  • Monoaromatic retinoids - etretinate, trans-acitretin, motretinide.
  • Polyaromatic retinoids - adapalene, tazarotene, tamibarotene, arotenoid methylsulfone.

In external medicinal and cosmetic products for the correction of aging, retinol, retinol palmitate, retinaldehyde, tretinoin, retinoic acid esters, isotretinoin are used, for the correction of photoaging - tretinoin, isotretinoin, arotinoid methylsulfonate, fenretinide, for the correction of acne - tretinoin, isotretinoin, motretinide, adapalene.

By the end of the 19th century. cytologists had almost exhaustive knowledge about the morphological side of mitosis. Further replenishment of data on cell division occurred mainly through the study of the most primitive organisms.

The process of division in prokaryotic (not having a formed nucleus) organisms (bacteria), which is genetically close to methylation (M. A. Peshkov, 1966), as well as mitosis in protozoa (I. B. Raikov, 1967), where they were found, were studied in detail extremely unique forms of this process. In higher organisms, the morphological study of mitosis proceeded mainly along the lines of studying this process in dynamics on living objects using microfilming. In this regard, the work of A. Bayer and J. Mole-Bayer (1956, 1961), performed on endosperm cells of some plants, was of great importance.

However, the vast majority of works of the 20th century. concerned the physiology of cell division, and it was in this section of the problem that the greatest success was achieved. In essence, the question of the causes and controlling factors of mitosis remained unexplored. The founder of this line of research was A. G. Gurvich.

Already in the monograph “Morphology and Biology of the Cell” (1904), Gurvich expressed the idea that there must be factors that determine the occurrence of mitosis, and they are most likely associated with the state of the cell itself that is beginning to divide. These still very general ideas were developed in a series of further studies by Gurvich, summarized in the monograph “The Problem of Cell Division from a Physiological Point of View” (1926). Gurvich's first important theoretical conclusion was the idea of ​​the dualism of factors that cause mitosis only when they are combined. One of these factors (or a group of factors) is associated with the endogenous processes of cell preparation for division (possibility or readiness factor). The other is exogenous to a given cell (implementation factor). Gurvich's further research was devoted mainly to the study of the second factor.

Experiments and theoretical considerations led Gurvich in 1923 to the discovery that most exothermic reactions both in the body and in vitro are accompanied by UV radiation. The most important biological consequence of this phenomenon was the stimulation of cell division, which is why these rays were called mitogenetic, i.e., causing mitoses. Over the following years, Gurvich (1948, 1959) and his colleagues carried out a large number of studies devoted to the problem of mitogenetic radiation. The stimulating effect of radiation has been elucidated on a wide variety of objects - from bacteria and yeast fungi to embryos and tissue culture cells of mammals (A. A. Gurvich, 1968).

In the first quarter of the 20th century. Data began to accumulate regarding the influence of external influences on mitosis - radiant energy, various chemicals, temperature, concentration of hydrogen ions, electric current, etc. Especially a lot of research was carried out on tissue culture. It has now been established that mitotic division is a consequence of a long chain of causes.

In contrast to early cytology, which focused on mitosis itself, modern cytology is much more interested in interphase. Using Gurvich’s terminology, we can say that now the study of the factors of readiness is in the foreground.

strength, ensuring the possibility of the cell entering into division.

This became possible thanks to new research methods, primarily thanks to autoradiography.

A. Howard and S. Pelk (1951) proposed dividing the entire mitotic cycle into four periods: postmitotic, or presynthetic (Gi); synthetic (S), during which DNA replication occurs; postsynthetic, or premitotic (G2); and finally mitosis (M). A large amount of factual material has been accumulated on the duration of individual periods and the entire mitotic cycle as a whole in a variety of organisms, normally and under the influence of various external and internal factors - radiant energy, viruses, hormones, etc.

A number of studies (M. Swann, 1957, 1958) are devoted to the energetics of cell division, and although many details remain unclear, it has become obvious that an important role in this regard belongs to high-energy compounds, in particular ATP. This substance not only participates in preparing the cell for division, but, according to G. Hoffmann-Berling (1959, 1960), is responsible for the mechanical processes underlying the divergence of chromosomes to the poles.

In elucidating the mechanism of the various stages of cell division, the works of the American researcher D. Mezius (1961), who studied various aspects of the physiology of mitosis, especially the role of the mitotic apparatus, which carries out the division process itself, played a particularly important role. Various ideas have been created about the mechanism of division of the cell body and about the physicochemical changes of cells during division. The study of chromosomes grew into an independent field of research, which turned out to be organically related to genetics and gave rise to cytogenetics.

Along with the study of individual mitoses, a significant number of studies were devoted to elucidating the patterns of mitotic activity of tissues, in particular, studying the dependence of cell proliferation on the physiological state of the body and the influence of various endogenous and exogenous factors.

The first studies of this nature were carried out on plant objects at the very beginning of the 20th century. in connection with the study of the periodicity of biological processes (A. Lewis, 1901; V. Kellycott, 1904). In the 1920s, a number of fundamental studies appeared on the daily rhythm of cell divisions in plant seedlings (R. Friesner, 1920; M. Stolfeld, 1921). In the 30-40s, a series of studies were carried out (A. Carleton, 1934; Ch. Blumenfeld, 1938, 1943; 3. Cooper, G. Franklin, 1940; G. Blumenthal, 1948; etc.), which studied mitotic activity in foci of cell reproduction in various laboratory animals. Significantly less such work has been carried out on the foci of human cell reproduction (3. Cooper, A. Schiff, 1938; A. Broders, V. Dublin, 1939; etc.).

In the USSR, the first study on the influence of physiological factors on the mitotic regime was published in 1947 by G. K. Khrushchov. Since the 50s, interest in the problem of the mitotic regime of the body has increased significantly (S. Ya. Zalkind, I. A. Utkin, 1951; S. Ya. Zalkind, 19.54, 1966; V. N. Dobrokhotov, 1963; I A. Alov, 1964; etc.). The daily rhythm of mitotic activity in mammals has been most fully studied.

The first attempts to analyze the mechanisms regulating mitotic activity were made in 1948 by the English researcher W. Bullough. Soviet cytologists (JI. Ya. Blyakher, 1954; I.A. Utkin, 1959; G.S. Strelin, V.V. Kozlov, 1959) paid great attention to the neurohumoral regulation of mitotic activity, establishing the reflex nature of the regulation of cell divisions. It turned out that the effect on the nervous system affects indirectly - through a shift in hormonal balance. It also turned out that the secretion of adrenaline, which inhibits mitotic activity, sharply increases. Removal of the adrenal glands leads to the switching off of the effect of inhibition of mitoses (A.K. Ryabukha, 1955, 1958). A number of studies are devoted to the study of the complex relationships between the mitotic and physiological activity of the organism (S. Ya. Zalkind, 1952; I. A. Alov, 1964).

Increasing interest in the problem of mitotic cycles and the widespread use of autoradiography has led to the fact that currently the vast majority of works are devoted to the study of the patterns of the mitotic cycle, analysis of the patterns of transition from one period to another, and the influence of various endogenous and exogenous factors on mitosis. This is undoubtedly one of the most promising directions in the study of the problem of cell proliferation (O. I. Epifanova, 1973).

Cytology of heredity

In the first half of the 20th century. In connection with the flourishing of genetics, cytological problems relating to heredity were intensively developed. This is how a new field of cytology arose - karyology.

The pioneer of karyological research was the Russian botanist

S. G. Navashin. Navashin can rightly be called the creator of cytogenetics; it is no coincidence that the first period in the development of this science is often called “Russian” or “Navashinsky”. Already in classical works on plant embryology, especially on the cytology of fertilization (1898), he focused his attention on the morphology of chromosomes in the cells of some lilies, in particular, horse hyacinth (Galtonia candicans). In 1916, Navashin published a work in which he gave a thorough description of the chromosome set of this plant. He was able to find on the chromosome (in the center or at its pole) a special uncolored region (which he called the “chromatic break”), now called the centromere or kinetochore, in the region of which the chromosome is attached to the spindle. Centromeres play an extremely important role in the process of chromosome splitting and their divergence to the poles of the dividing cell. Navashin was the first to show that the structure of chromosomes is not at all immutable, but is subject to changes in phylogenesis and under certain special conditions of existence (for example, in seed cells during long-term storage). Using a number of plant objects (Crepis, Vicia, Muscari, etc.), Navashin’s students showed that karyolotic analysis can be used for phylogenetic inferences. Somewhat later, karyological studies began on animal and human cells. Navashin also participated in these works. After his death, in 1936, a work was published on the reduction (diminution) of chromatin during the development of the horse roundworm egg, which confirmed the conclusions of T. Boveri (1910).

Detailed karyological work was carried out in the 20-30s by the Soviet cytologist P.I. Zhivago. He and his collaborators studied the karyotype of domestic birds (chickens, turkeys; 1924, 1928), small cattle (1930) and humans (1932). Zhivago not only identified a number of karyotypes, but also began to explore the question of the constancy of the number of chromosomes within one organism. Based on literary data (on Diptera) and studies of a number of objects (emus, rheas, humans), Zhivago (1934) came to the conclusion that significant fluctuations in the number of chromosomes are observed in individual cells and entire tissues (especially in embryos). He attached great importance to these differences, since they lead to changes in the genome, and, consequently, in the hereditary properties of the organism. He also suggested that the presence of cells with different numbers of chromosomes may have adaptive significance, since it increases the possible variants of karyotypes for subsequent selection. This point of view, expressed over 30 years ago, is currently shared by many researchers.

A major role in the development of this direction was played by K. Belar’s ​​book “Cytological Foundations of Heredity” (1928, Russian translation 1934). The section devoted to the connection of chromosomes with heredity is preceded by cytological chapters themselves, containing data on the structure of the nucleus and cytoplasm, cell division, fertilization and maturation of germ cells, and parthenogenesis. The structure of chromosomes not only in higher vertebrates, but also in invertebrates, protozoa and plants is examined in great detail and in a comparative aspect. Contains valuable data regarding the individuality and variability of chromosomes, the exchange of fragments during crossing over, chromatin diminution, and the pathology of mitosis. Belar's book remained for a long time the best monograph on the cytology of heredity.

Gradually, due to the intensive development of genetics, the cytology of heredity turned into cytogenetics, the history of which is briefly outlined along with the history of genetics (see Chapters 13 and 24). In the second half of the 20th century. Several completely new, very promising areas of research have emerged.

First of all, we should mention cytoecology, which studies the role of the cellular level of organization in the adaptation of the organism to environmental conditions. In the USSR, this direction, closely related to the biochemistry of the cell and especially to the study of the properties of cellular proteins, was widely developed in the works of V. Ya. Aleksandrov and B. P. Ushakov.

Over the past 10-20 years, much attention has been drawn to the study of the general physiology of the cell and, in particular, the patterns of synthesis and consumption of substances, both those involved in the main life processes and those that are its specific products (secrets). This same range of issues includes the study of restoration processes in the cell, i.e. physiological regeneration, which ensures the restoration of destroyed or lost cellular structures and substances and takes place at the molecular level.

The problems of determination, differentiation and dedifferentiation of cells have acquired great importance in cytology. They play an important role in embryonic cells and various categories of cells cultured outside the body (A. De-Rijk, J. Knight, 1967; S. Ya. Zalkind, G. B. Yurovskaya, 1970).

Cytopathology constituted a unique section of cytology - an area bordering on general pathology and which made significant progress in the last decades of the 20th century. The term “cytopathology” is used to designate a branch of biology in which the study of general pathological processes is carried out at the cellular level, and as a system of knowledge about pathological changes in an individual cell. As for the first direction, after the classical works of R. Virchow, attempts to reduce the essence of the pathological process to changes in microscopic and submicroscopic structures were made repeatedly. Many examples of such use of cytological analysis to understand pathological processes in the body are contained in the works of R. Cameron (1956, 1959).

The second direction can be considered as purely cytological. It aims to study the pathology of the cell itself and its organelles, i.e., morphological, biochemical and physiological deviations from the norm observed during various pathological processes occurring in the cell, regardless of their effect on the state of the tissue, organ or the entire organism. The development of this direction is associated primarily with the accumulation of data on changes in cells that occur as a result of their natural aging, as well as various sudden cytopathological changes observed under the influence of certain unfavorable factors (physical, chemical, biological) of the external environment. Particularly significant development has been achieved in the study of pathological changes under the influence of adverse effects on the cell in experiment and the study of the mechanism of action of such factors. These studies have been widely developed, primarily in radiobiology, where a comprehensive study of the cell response to the effects of radiant energy is possible not only at the cellular or subcellular, but also at the molecular level.

It is known that some cells continuously divide, for example bone marrow stem cells, cells of the granular layer of the epidermis, epithelial cells of the intestinal mucosa; others, including smooth muscle, may not divide for several years, and some cells, such as neurons and striated muscle fibers, are not able to divide at all (except in the prenatal period).

In some tissue deficiency of cell mass eliminated by rapid division of the remaining cells. Thus, in some animals, after surgical removal of 7/8 of the liver, its weight is restored almost to its original level due to cell division in the remaining 1/8. Many glandular cells and most cells of the bone marrow, subcutaneous tissue, intestinal epithelium and other tissues, with the exception of highly differentiated muscle and nerve cells, have this property.

Little is known yet how the body maintains the necessary number of cells of different types. However, experimental data suggest the existence of three mechanisms for regulating cell growth.

Firstly, division of many types of cells is under the control of growth factors produced by other cells. Some of these factors come to cells from the blood, others from nearby tissues. Thus, the epithelial cells of some glands, such as the pancreas, cannot divide without a growth factor produced by the underlying connective tissue.

Secondly, most normal cells stop dividing when there is not enough space for new cells. This can be observed in cell cultures, in which cells divide until they come into contact with each other, then they stop dividing.

Thirdly, many fabric crops stop growing, if even a small amount of the substances they produce gets into the culture liquid. All of these cell growth control mechanisms can be considered variants of the negative feedback mechanism.

Regulation of cell size. The size of a cell depends mainly on the amount of functioning DNA. Thus, in the absence of DNA replication, the cell grows until it reaches a certain volume, after which its growth stops. If you use colchicine to block the process of spindle formation, you can stop mitosis, although DNA replication will continue. This will lead to the amount of DNA in the nucleus significantly exceeding normal, and the volume of the cell will increase. It is assumed that excessive cell growth in this case is due to increased production of RNA and protein.

Differentiation of cells in tissues

One of growth characteristics and cell division is their differentiation, which is understood as a change in their physical and functional properties during embryogenesis with the aim of forming specialized organs and tissues of the body. Let's look at an interesting experiment that helps explain this process.

If from eggs If you remove the nucleus of a frog using a special technique and replace it with the nucleus of a cell of the intestinal mucosa, then a normal frog can grow from such an egg. This experiment shows that even such highly differentiated cells as those of the intestinal mucosa contain all the necessary genetic information for the development of a normal frog organism.

It is clear from the experiment that differentiation occurs not due to gene loss, but due to selective repression of operons. Indeed, in electron micrographs one can see that some DNA segments “packed” around histones are condensed so strongly that they can no longer be unwoven and used as a template for RNA transcription. This phenomenon can be explained as follows: at a certain stage of differentiation, the cellular genome begins to synthesize regulatory proteins that irreversibly repress certain groups of genes, so these genes remain inactivated forever. Be that as it may, mature cells of the human body are capable of synthesizing only 8,000-10,000 different proteins, although if all genes were functioning, this figure would be about 30,000.

Experiments on embryos show that some cells are able to control the differentiation of neighboring cells. Thus, the chordomesoderm is called the primary organizer of the embryo, since all other tissues of the embryo begin to differentiate around it. Transforming during differentiation into segmented dorsal mesoderm consisting of somites, chordomesoderm becomes an inducer for surrounding tissues, triggering the formation of almost all organs from them.

As another example of induction development of the lens can be cited. When the optic vesicle comes into contact with the head ectoderm, it begins to thicken, gradually turning into the lens placode, which, in turn, forms an invagination, from which the lens is eventually formed. Thus, the development of the embryo is largely due to induction, the essence of which is that one part of the embryo causes the differentiation of another, and that causes the differentiation of the remaining parts.
So although cell differentiation in general still remains a mystery to us, many of the regulatory mechanisms that underlie it are already known to us.

If we characterize the known phytohormones in the most general terms, then we can say that the distinctive feature of auxins is the stimulation of cell elongation, gibberellins - stimulation of stem growth, and kinins are characterized by their ability to cause cell division in tissues that are not responsive to other influences under optimal nutritional conditions.

That is, kinins can be called cell division hormones.

However, the physiological spectrum of action of kinins is somewhat wider and is not limited only to division. They also influence cell elongation and differentiation and other processes. It should be noted that kinins exhibit their activity only in the presence of auxins. For example, in the root callus formation test, the activity of quinines is closely related and depends on the interaction with auxin, and both groups of hormones cause callus growth: auxins - an increase in size, kinins - their division. Normal growth is determined by the balance between them.

Many researchers have repeatedly noted the effect of quinines on root growth. In this case, both inhibition and stimulation of cell division and elongation were observed. Inhibition occurred at high concentrations of hormones, and stimulation depended on the experimental conditions and the physiological state of the research object.

The growth of bean leaf discs and the germination of lettuce seeds is stimulated by quinines and red light and inhibited by far-red light. However, according to Miller, kinins cannot completely replace red light, since they do not take part in the photoreaction and have a mechanism of action different from red light.

All of the above diversity of action of kinins was studied in the vast majority of cases using one representative of this class of growth hormones - kinetin. Actually, kinetin cannot be called a real hormone, since this substance is not isolated from higher plants.

In its chemically pure form, kinetin was first isolated from yeast extract and herring sperm by a group of employees at the University of Wisconsin, USA in 1955. They also established the structure of this compound, which is 6-furfurylaminopurine. Somewhat later, in 1957, Skoog et al. isolated kinetin from old or autoclaved DNA preparations. A year later, a report of the chemical synthesis of kinetin appeared.

A synthetic study of chemical analogues of kinetin showed that the adenyl part of the molecule plays the main role in the manifestation of the property of high biological activity, while the furfuryl side chain can be replaced by other non-polar groups. Using various variants of this substitution, about 30 highly active and an even larger number of less active compounds were obtained. These compounds were given the group name “kinins,” which later began to be applied to substances found in extracts from higher plants that activate cell division, like kinetin. Substances that strongly stimulate cell division have been found in plant extracts from the liquid endosperm of coconut, endosperm of corn, from developing parthenocarpic banana fruits and unripe horse chestnut fruits, tobacco and carrot leaves, from grapes, tumor tissue of crown galls, female gametophyte of ginkgo and many others.

Using cell division as a screening test, different researchers in three different laboratories isolated kinins from liquid corn endosperm extract. However, the quantities of the obtained drugs are not sufficient for their complete chemical identification. They all agree that kinins are derivatives of adenine, unsubstituted, with the exception of the nitrogen atom in the sixth position. In all cases, the isolated substance was able to produce only part of the cell division stimulating activity.

A further comparison of the properties of active preparations from coconut endosperm and corn endosperm purified on ion exchange resins casts doubt on the fact that the compound found is truly native kinin, although this test is not without methodological doubts.

The final elucidation of the chemical nature of native kinins is a matter of time, since methods for their isolation have already been largely developed. This is also supported by the relatively easy spontaneous formation of kinetin from DNA.

The lack of knowledge about the exact chemical nature of native kinins limits studies on their biogenesis and transformations in plant tissues. Kietin-like molecules are involved in metabolism in plants along the normal pathway for purinosis. The weak mobility of kietin within plant tissues indicates that native kinins may be synthesized by cells that require it.

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