Chapter 1.

Introduction to Cell and Molecular Biology

1.1 Definition. Object of study.

Cell and Molecular Biology is a branch of biological sciences that studies structure and functions of the cell as basic unit of living organisms. Cell and Molecular Biologys object of study is an ideal cell displaying common (general) features of all cells. Specialized cells like neurons, epithelial cells, muscle fibres etc. are studied by histology.

1.2 Evolution of knowledge about cells The first steps in progress of knowledge concerning cells were made in the 16th century when the first microscopes were assembled. Discovery of cells belongs to the English Robert Hooke (1665). While was studying a thin slice of cork, he observed an image as a honey comb and designated those cavities as cells (in Latin, cella = room, cellula = small room). He saw in fact cellulose walls separating the former vegetal cells, as in cork there are no living cells. The first living cells were discovered in 1674 by the Dutch Anthony van Leewenhoek: protozoa in water, bacteria (opening the new field of bacteriology), red and white blood cells, or spermatozoa (he also was the first to describe cellular movements). From this point knowledge concerning the cells evolved on two main paths: the first path was of techniques and methods used for cell study; the second one was of conceptual progresses. Concerning the first path, for over 150 years the light microscopy was the only method available for cell study. During the 19th century this method was improved, and thus the first conceptual progresses (known as generalizations) were made. The first generalization belongs to Robert Brown; he showed that nucleus was a common (constant) component of all cells. As we shall see later, this is true only for eukaryotic cells, with a real nucleus, and not for prokaryotic cells (without nucleus). The second generalization consisted in a primitive concept describing the cell as a mass of living matter (called protoplasm) containing a nucleus (in which the nucleolus could be observed) and cytoplasm (not to be confounded with the protoplasm). At its periphery, the cell has a membrane called plasmalemma (that cannot be observed using a light microscope).
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The most significant generalization of that time was the cell theory of the German scientists Schleiden (botanist) and Schwann (zoologist). In its classical form, this theory contained three ideas: a) body of a living organism is composed of cells and substances produced by cells; b) cells have their own life; c) cells life is subordinated to life of entire organism. Later on, these three ideas were considered as a first thesis of the modern cell theory that also included two other theses: 2. All cells arise from pre-existent cells (they are not assembled from non-living matter); the original Latin formulation of Rudolf Virchow was: omnis cellula e cellula. 3. All cells that build a pluricellular organism derive from a single cell, egg cell, formed in turn by fusion of two cells (gametes): spermatozoon and ovule in case of the sexuate reproduction; next the pluricellular organism (like human body) is formed by growth and multiplication of the egg cell. During the last decades the later thesis was modified by developing of cloning as an alternative, non-sexuate reproduction of organisms. As result of these progresses, a new branch of biological sciences was born towards the end of the 19th century: classical cytology that studied the cells based on observations with light microscopes. The studies were conducted mainly on fixed and coloured cells (dead cells) and lesser on living cells. This is why the classical cytology had a morphologic character. In the second half of the 19th century, several new processes (or phenomena) and new cell components were described. Thus, cell division was discovered, which, in the case of eukaryotic cells is an indirect division with two forms: mitosis and meiosis. During the division, chromosomes (in Greek: chroma = colour, soma = body) were observed. Later on, several cytoplasmic organelles were discovered within the cytoplasm: cellular centre (centrosome), visible during the cell division, mitochondria, Golgi apparatus, and also cytoplasmic structures based on endoplasmic reticulum (the Berg bodies, Nissl granules or the ergastoplasm). In parallel, several studies were oriented towards the functional aspects of cells, leading to a separated chapter of Cellular Physiology in a Physiology book in the 19th century. On the other hand, Rudolph Virchow set the basis of Histopathology by describing in his book Cellular Pathology microscopic modifications at cellular level in humans deceased by different diseases. He also was the first to apply the knowledge concerning cells in medicine. During the 20th century, several significant progresses were made both at conceptual level and in the field of investigation methods and techniques. Hereby, methods for study the living
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cells were developed: tissue cultures and, then cell cultures; cell microsurgery: penetration of plasmalemma was performed (avoiding cell death), penetration of nuclear envelope (resulting in cell death), or injection of various substances inside the cell. This method, also known as micromanipulation, has now important medical applications, such as in vitro fertilization. But first among techniques that improved the study of cells in the 20th century was electron microscopy, perfected towards mid-century. By conferring not only a higher magnification of images but a higher resolution as well, this technique made the transition from the classical cytology to the modern cytology. Several important progresses due to electron microscopy can be mentioned: - existence of a cell membrane at the cell periphery was proven; - discovery of new intracellular structures and organelles: description of endoplasmic reticulum, peroxisomes or of cytoplasmic differentiations (as filaments and microtubules); - existence of Golgi apparatus was confirmed; - ultrastructure (term implying aspects visible in electron microscopy) of cell organelles (mitochondria, Golgi apparatus, peroxisomes) was also solved. There are many techniques of electron microscopy, among which we can mention: transmission electron microscopy, scanning electron microscopy or freeze-fracturing. George Emil Palade (the first Romanian distinguished with the Nobel Prize, in 1974, for Medicine or Physiology) had fundamental contributions in the development of electron microscopy: he elaborated and perfected the technology for ultrathin sections, introduced the osmium tetraoxide as a fixative and had various other contributions that will be mentioned later on during the lectures. Palade also essentially contributed to the development of a second technique that improved the study of cells: a biochemical technique known as cell fractioning by differential centrifugation. Palade introduced saccharose as homogenization medium and this way, living components of the cell were isolated. Using this technique, the Belgian Christian de Duve discovered lysosomes. These two techniques produced a new revolution in the cell study: transition from the modern cytology to cell biology (around 1960). The morphological and biochemical data converged in order to study the cellular structures from a structural point of view and the biochemical processes occurring at each level.
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On the other hand, speaking about the conceptual progresses, cell biology was born through a fusion of all knowledge of cytology, histology, cell physiology, cell biochemistry and biophysics, microbiology, virology and genetics. The next revolution in the cell study, the transition from cell biology to cell and molecular biology (around 1975), was given first of all by physical and biochemical techniques of molecular biology: X-ray diffraction (by which were specified the inter atomic distances and also the three-dimensional structures for macromolecules such as nucleic acids and proteins), methods with neutrons, nuclear magnetic resonance (NMR), electronic spin resonance (ESR), study of DNA sequences, or study of secondary, tertiary and quaternary structure of proteins and nucleic acids. From a conceptual point of view, molecularisation of cell biology was accompanied by new concept of molecular biology: study of living matter through relationship between structure and functions at molecular level. If initially it was applied to nucleic acids and proteins and next to molecular study of genetic material (molecular genetics), nowadays this concept is applied to the entire living matter, leading thus to molecular biology of membranes, of chromatin, of mitochondria, of bacteria, etc. Moreover, the study of cell was revolutionized after 1975 when complex biotechnologies were developed: biotechnology of recombinant DNA, biotechnology of producing monoclonal anti-bodies, etc.

1.3 Current definition of cell and molecular biology

Cell and molecular biology is the branch of biological sciences that studies the cell structure and functions in a complex and unitary way, starting from aspects visible in light microscope (morphology), to aspects visible in electron microscope, going down at molecular level in profoundness of each cellular compound. Finally, image of the cell is reconstituted in all its complexity, and the cell is integrated in higher levels of organization of the living matter. In pluricellular organisms the integration levels of the living matter are represented by cell, tissue, organ, apparatus or system, and finally the whole organism. Above organisms there are two more superior levels of integration of the living matter, represented by biocenosis (all organisms living in a given area) and biosphere (all organisms at planetary level).
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1.4 Importance of cell and molecular biology for medicine

For a medical student, the knowledge concerning the cells is indispensable for understanding data obtained in fundamental biomedical disciplines (physiology, histology, biochemistry, biophysics, genetics, microbiology, virology), in those that make connections between fundamental and clinic (pharmacology, physiopathology, immunology), and in clinical ones. Until the 19th century morpho-clinical medicine was developed, while in the first half of the 20th century, physiopathological and biochemical directions were developed. Starting with the second half of the 20th century the development of medicine has been based upon the knowledge about cells. First of all, is obvious that if the human organism is made of cells, any disease is manifested at cellular and molecular level. Thus it is also very clear that etiopathogenesis of diseases must be studied down to fine modifications at molecular level (such as localization of molecular defect in certain diseases, diagnosis and monitoring of some diseases using specific molecules), including diagnosis in a presymptomatic state of diseases, and in some diseases (genetic diseases) even prenatal diagnosis can be established. Modern treatment of diseases cannot be performed without knowing the cellular and molecular mechanisms of action of drugs and of other therapeutic methods (radiotherapy, physiotherapy, psychotherapy, etc.). Moreover, individualized pharmacological treatment is important, according to genetic features of every patient, as genetic therapy is of actuality. The cellular and molecular medicine is applied not only to laboratory lines or to those of internal medicine (cardiology, neurology, pediatrics, immunology), but also to surgical ones, the practice of modern surgery (including transplants) being inconceivable without cognition and direct application of knowledge of cell and molecular biology.

1.5 Romanian contributions to the development of cell and molecular biology

It is a proud for us to mention several of the great Romanian scientists whose discoveries related to cells will be forever Romanian contributions to the world science. Gheorghe Marinescu (neurologist) had important contribution in neurocytology, and published in Paris, in 1909 The nervous cell, a true Bible of neurobiology for many decades. Victor Babe discovered the passive immunization (fundamental of serotherapy), for which he deserved to be awarded with the Nobel Prize for Physiology or Medicine in 1901 (the Prize was awarded to the German von Behring). Babe made also other discoveries:
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discovered an entire class of pathogen parasites (over 40 new microorganisms) named Babesia in his honour, parasites responsible for producing diseases called babesiosis in animals and also in humans that come in contact with animals;

discovered characteristic corpuscles in nervous cells of animals and humans deceased due to infections with rabies. Because these corpuscles were independent discovered by the Italian Negri, they were named the Babes-Negri corpuscles.

discovered the Babe -Ernst bodies in diphtheria bacillus (also discovered by the German Ernst). He was the first that foresaw that between microorganisms antagonistic relationships are possible (antibiosis). Victor Babe published the first textbook of microbiology, in Paris in 1886. Ion Cantacuzino had contributions in comparative immunology, microbiology,

experimental medicine, and discovered stimulator factor of cell secretion in different biological liquids. He founded the Institute of sera and vaccines in Bucharest, later named Cantacuzino Institute. His activity made Romania the second country (after France) that introduced preventive vaccination against tuberculosis. George Emil Palade was awarded in 1974 with the Nobel Prize for Physiology or Medicine, together with the Belgians Albert Claude (who introduced the technique of cell fractioning by centrifugal differentiation) and Christian de Duve (discoverer of lysosomes). Besides his contributions already presented in Chap. 1.2., other important discoveries can be mentioned here: discovered ribosomes (the Palades granules) and specified their role in protein synthesis; described ultrastructure of mitochondria (mitochondrial cristae) explained the way of cell secretion described vesicles transport in capillary endothelium (known today as transcytosis). described recycling process of cell membranes and also aspects of cell membranes biogenesis.

Chapter 2. General notions concerning the cells

2.1 Prokaryote and eukaryote cells

There are two major categories of cells: prokaryotes (such as bacteria or green-blue algae) and eukaryotes (in plants, animals and humans). The prokaryotes are always unicellular organisms. The eukaryotes can be unicellular (protozoa) or pluricellular organisms (metazoa animals & humans, metaphyta plants). There are five essential differences between the prokaryote and the eukaryote cells concerning their cellular organization: 1. Eukaryotes from the Greek (karyon = nucleus) are cells with a proper nucleus, surrounded by a nuclear envelope, and containing a specific number of chromosomes. Prokaryotes dont have a real nucleus, but rather a nuclear material organized in a structure called nucleoid (nucleuslike), in direct contact with cytoplasm. This nucleoid consists of a single DNA molecule unique chromosome of prokaryotes, being in a tightly packed state. 2. Prokaryotes multiply by direct division (or binary fission), resulting in two identical cells. Eukaryotes have a more complex multiplying process indirect division, with two forms: mitosis and meiosis. During this process, chromatin (nuclear material chromosomes are made of) condenses and chromosomes become visible as sticks. 3. Eukaryotes have several cytoplasmic organelles in cytoplasm (mitochondria, lysosomes, peroxisomes, Golgi apparatus) separated by organelle membranes from the rest of the cytoplasm. Thus, the cytoplasm of eukaryote cells is partitioned, having spaces of definite composition, in which specific enzymatic processes occur in other conditions than those in the cytoplasm. Prokaryotes dont have cell organelles separated by membranes, and dont display a cytoplasm partitioning. 4. Besides the plasma membrane (plasmalemma), prokaryotes also have on their outer side a cell wall containing a specific marker: the N-acetyl muramic acid (from the Greek muros = wall). The plasmalemma can exhibit cytoplasmic extensions, called mesosomes. Eukaryotes dont have such structures; and if the plants do have cell walls, this is made of cellulose and very different as compared to the prokaryotic structure. 5. In the eukaryote cell the so-called cytoplasmic differentiations (filaments, and microtubules) were described; they form the cytoskeleton, and are responsible for the cellular movements specific to eukaryotes, such as the amoeboidal locomotion or the cytoplasmic streaming. In the
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case of prokaryote cells, locomotion is provided by relatively simple structures called flagella very different as compared to flagella of some eukaryotes (in the human body, the only cell with flagellum is spermatozoon). It should be noted that there are various other differences between the prokaryote and eukaryote cells, which will be later mentioned (in biosynthesis of nucleic acids and proteins). In the cytoplasm of vegetal cells some specific structures can be described: large cytoplasmatic vacuoles (that absorb water from the cytoplasm, contributing to the reduction of the cytoplasmatic osmotic pressure), and chloroplasts (organelles that contain chlorophyll and produce the photosynthesis). In animal cells the cytoplasmatic vacuoles are found only in pathologic conditions. A special notice should be given to viruses. They can be defined as biological entities halfway between alive and dead matter. The viruses have a simple structure: a core of a nucleic acid (genetic material of the virus, either DNA or RNA) surrounded by identical protein subunits (arranged in a certain spatial symmetry) that form a capsid. Some viruses also possess an envelope (in HIV, for instance, which is rapidly changing its membrane antigenic composition, process that makes production of vaccines more difficult). On the scale of evolution, as far as we know from present scientific evidences, the earth was born some 4.5-5 billion years ago; the prokaryotes appeared about 3.5 billion years ago and the eukaryotes 1.5 billion years ago, while man, around 1.8 million years.

2.2 Number, shape and size of eukaryote cells

Human body is made up of an extremely high number of cells, the exact amount of which is difficult to appreciate, but is in the range of millions of billions (1015). The most numerous are red blood cells, in range of thousands of billions (1012), of which about 10 million die daily and are replaced with young cells from hematopoietic bone marrow. Hepatocytes, and also neurons, are in range of hundreds of billions (1011), of which several millions die daily, while glial cells are 10 times more numerous (1012). The cells have a large variety of shapes, depending on their age and specific role they have to accomplish. The young cells are generally spherical (ovule, pluripotent stem cell etc.). As they mature and multiply, their differentiation is governed by the law of adjusting the shape to function. For instance, the cells with contractile roles are elongated (muscular tissues are spindled), those responsible for conductibility have prolongations (neurons), while the red blood
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cells take the shape of a biconcave disc, thus producing the largest possible exchange area for a given volume in order to maximize oxygen transfer towards tissues. The glial cells are starshaped, while others are cubic, cylindrical or polyedrical (endothelial cells). Some ones have rather peculiar shapes, as is the case of the Purkinje cells in the cerebellum. Mean sizes of human cells are in range of 20-30 m (1 m = 10-6 m). The smallest are neurons from cerebellum (3-6 m in diameter) and lymphocytes (4-5 m). The largest are giant neurons from frontal cortex (125-150 m, of pyramidal shape), and the ovocyte about 250 m (note that yolk of ostrich egg is a cell of about 10 cm in diameter). Volume of cells may vary from 300 to 15.000 m3. In connection with this must be mentioned the law of volume constancy: certain types of cells, from a certain organ, have approximately the same volume in various animal species regardless of the body size. As an example, the red blood cells are of about 6 m in diameter in mice, 8 m in humans, and 9 m in elephants, thus there is a very little variance between the sizes of these cells at different species, compared to enormous differences in sizes of organisms from these species. Also, while the liver has very different sizes for mouse, man or elephant, the hepatocytes diameter differs insignificantly from one species to the other. To conclude, the dimension of various organs is given not by the size of the cells that compose the organ, but by the number of cells of that organ.