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Biology : Is a science that studies living things and provides an under standing of life. Q : How to define life 1.Living things are organized. 2.Take.

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Presentation on theme: "Biology : Is a science that studies living things and provides an under standing of life. Q : How to define life 1.Living things are organized. 2.Take."— Presentation transcript:

1 Biology : Is a science that studies living things and provides an under standing of life. Q : How to define life 1.Living things are organized. 2.Take materials and energy from the environment respond to stimuli. 3.Living thing produce and develop. 4.Living thing have adaptation.

2 1. Living things are organized. - Cells are makeup of molecules that contain atom. Which are the smallest units of matter that can enter into chemical combination. - In multi cellular organisms, similar cells combine to form a tissue. - Tissues makeup organs, in turn work together in system.

3 2. Living thing take materials and energy from the environment respond to stimuli. - Living thing find energy and nutrients by interacting with their surroundings. - The ability to respond often results in movement. - The ability to respond helps ensure survival of the organism and allows it to carry on its daily-activities. - All together, we call these activities the behavior of the organism.

4 3. Living thing produce and develop. - Life comes only from life. Every type of living thing can reproduce or make anther organism. - There are two general methods of reproduction:- a sexual: without the formation of specialized sex cells. Sexual: which distinct sex cells are produced.

5 4. Living thing have adaptation. - Living things are capable of surviving in great variety of environments. - The term adaptations is used in two senses. a- Short term changes in response to change in the environment. b- The long term major changes resulting from genetic differences and production of new varieties.

6 Cations: Calcium ions, Ca 2+, are the most abundant cations (positive ions) in the body, making up about 1.5% of total body weight. About 99% is found in bones and teeth, largely in combination. They combine with phosphate ions to form calcium phosphate which increases the rigidity and hardness of bones and the enamel in teeth. Calcium ions are also involved in blood clotting, normal muscle contraction and nerve activity.

7 Sodium ions, Na +, are the main cations in extracellular fluids. They affect the transport of water through cell membranes by osmosis. They are also part of the hydrogencarbonate buffer system. Potassium ions, k +, are the main cations in intracellular fluids. They contribute to the transmission of nerve impulses and muscle contraction. Magnesium ions, Mg 2+, are important because of their role in the normal functioning of muscle and nerve tissue, bone formation and as a component of many coenzymes. A normal diet provides sufficient quantities of calcium, sodium, potassium and magnesium ions.

8 Anions: Anions are negative ions. Chloride ions, Cl -, are important in the acid-base balance of blood and the water balance of the body, and in the formation of hydrochloric acid in the stomach. They are found in intracellular and extracellular fluids. Phosphate ions, PO 4 3-, have more roles than any other inorganic ions in mammals. They are important for the formation of bones and teeth, as a buffer in blood, their role in muscle contraction and nerve impulses, as a component of many coenzymes, for their role in transfer and storage of energy in ATP, and as a component of DNA and RNA.

9 Our food consists of chemicals that are necessary to :- necessary to :- 1\ Provide energy. 2\ Rerpair tissue. 3\ Regulate body machine.

10 Water is essential to life :- The existence of living things is dependent on the chemical and physical characteristics of the chemical and physical characteristics of water :- water :- 1\ The temperature of liquid water rises and falls more slowly than of most other liquids. more slowly than of most other liquids. 2\ Water has a high heat of vaporization. 3\ Water is universal solvent and facilitates chemical reaction both outside and with in living chemical reaction both outside and with in living systems. systems. 4\ Water has a high surface tension.

11 5\ Unlike most substances frozen is less dense than liquid water. 6\ Water molecules are cohesive and adhesive. 7\ Water accounting 70% or more of total cell mass. 8\ The critical property of water is that it is a polar molecule, in which the hydrogen atoms have a slight positive charge and the oxygen has a slight negative charge. Because of their polar nature, water molecules can from hydrogen bonds with each other or with other polar molecules as well as interacting with positively or negatively charged ions. As a result of these interactions, ions and polar molecules are readily soluble in water ( Hydrophilic )

12 In contrast, non polar molecules, which cannot interact with water, are poorly soluble in an interact with water, are poorly soluble in an aquous environment ( Hydrophobic ). aquous environment ( Hydrophobic ). When water ionizes, it releases an equal number of hydrogen ( H + ) and hydrogen ions ( OH - ) :- of hydrogen ( H + ) and hydrogen ions ( OH - ) :- H – O – H H + + OH -

13 Acids and Basses Acids ( High H + concentrantion ):- Are molecules that dissociate in water, releasing hydrogen ions (H + ), so called releasing hydrogen ions (H + ), so called proton donor. proton donor. For example, an improvtant inorganic acid is hydrochloric acid ( HCL ), which dissociation in this manner :- HCl H + + CL - HCl H + + CL -

14 Dissociation is almost complete ; therefore, this is called a strong acid. If hydrochloric acid is is called a strong acid. If hydrochloric acid is added to a breaker of water, the number of added to a breaker of water, the number of hydrogen ions ( H + ) increases hydrogen ions ( H + ) increases Bases ( Low H + concentration ) Are molecules that either take up hydrogen ions (H + ) or release hydroxide ions (OH - ) ( accept protons ) for example, an important (H + ) or release hydroxide ions (OH - ) ( accept protons ) for example, an important inorganic base is sodium hydroxide ( NaOH ), inorganic base is sodium hydroxide ( NaOH ), which dissociates in this manners which dissociates in this manners NaOH Na + + OH -

15 Dissociation is almost complete, therefore,sodium hydroxide is called a strong base. If sodium hydroxide is added a beaker of water the number of hydroxide ions in creases. sodium hydroxide is added a beaker of water the number of hydroxide ions in creases. PH Scale :- The PH scale is used to indicate the acidity and alkalinity of a solution.A pH of exactly 7 is neutral pH. Pure water has an equal number of hydrogen ions ( H + ) and hydroxide ions ( OH - ).

16 The pH scale ranges from 0 to 14. A pH of 7 has an equal concentration of hydrogen ions ( H + ) and hydroxide ions ( OH - ). Above pH 7 there are more hydroxide ions, and below pH 7 there are more hydrogen ions than hydroxide ions.

17 0.0 Hydrochloric acid 1.0 Stomach acid 5.6 Normal rain water 6.0 Urine 6.5 Saliva 7.0 Pure water, tears 7.4 Human blood 8.0 Sea water 14 Sodium hydroxide

18 Buffers keep pH steady  The pH of our blood when we are healthy is always about 7.4, that is just slightly basic (Alkaline).  pH stability is possible because the body has built in mechanism to prevent pH changes.  Buffers are the most important of these mechanisms.  Buffers help keep the pH with in normal limits because they are chemicals or combinations of chemicals that take up excess hydrogen ions (H+) of hydroxide ions (OH-). for example :- ♦Carbonic acid CH 2 CO 3 is a weak acid that minimally dissociates and then reforms in the following manner :- H 2 CO 3 H + + HCO 3 -

19 ♦Blood always contains some carbonic acid and some bicarbonate ions. When hydrogen ions (H + ) are added to blood, the following reaction occurs :- H + + HCO 3 - H 2 CO 3 ♦When hydroxide ions (OH-) are added to blood this reaction occurs :- OH - + H 2 CO 3 HCO 3 - + H 2 O These reactions prevent any significant change in blood pH.

20 Cell Biology Cell biology : is the specialized branch of biology which deals with the study of structure and function of cell organelles. Cytology :- is the science that deals with studies the cell. Cell differ markedly in :- 1.structure according to their location and activities, because they have different job to do, for example (a cell from a piece of skin is very different from a cell taken from a piece of bone). 2.Cells also have a variable from shape ; for example (the white blood cell (WBC) in human, other cell e.g. egg cells, sperm cells, nerve cells.

21 3.Size : e.g. egg cells can be seen with the naked eye, but the nerve cells can be seen by the microscope. Every cells is made up of :-  Cytoplasm : the living material in which various activities take place and which many organelles are found.  Plasma membrane :- surrounded the cytoplasm.  Nucleus :- contain the genetic material.

22 Cell are divided into two classes :- 1.Prokaryotic cells : for example, bacteria (Bacilli, cocci, spirillum) it's characters :-  prokaryotic cells are generally smaller and simpler than eukaryotic cells the size about 1-5 mm.  prokaryotic cells (bacteria) lack nuclear envelope.  in addition to the absence of a nucleus, their genomes are less complex.  they do not contain cytoplasmic organelles or a cytoskeleton.  prokaryotic cells have no histone protein bound to the DNA.  prokaryotic cells divide by (Binary fission) and they contain ribosome (70s).


24 2.Eukaryotic cells :- for example amoeba, paramecium.  eukaryotic cells are larger and more complex than prokaryotic cells.  the largest and most prominent organelles of eukaryotic cells is the nucleus, with a diameter of approximately 5 mm, which is surrounded by nuclear membrane, the nucleolus is also present.  The DNA is combined with histone protein.  eukaryotic contain with a variety of membrane enclosed organelles with in their cytoplasm.  They contain ribosome (80s)  Eukaryotic cells divided by either mitosis and meiosis.


26 Summary of the Differences Between Prokaryotic and Eukaryotic Cells Eukaryotic cells Prokaryotic Cells larger cells (> 10 mm)small cells (< 5 mm) often multicellularalways unicellular always have nucleus and other membrane-bound organelles no nucleus or any membrane-bound organelles, such as mitochondria DNA is linear and associated with proteins to form chromatin DNA is circular, without proteins ribosomes are large (80S)ribosomes are small (70S) always has a cytoskeletonno cytoskeleton motility by flexible waving undulipodium, made of tubulin motility by rigid rotating flagellum made of flagellin cell division is by mitosis or meiosiscell division is by binary fission reproduction is asexual or sexualreproduction is always asexual common metabolic pathwayshuge variety of metabolic pathways

27 Endosymbiosis Prokaryotic cells are far older and more diverse than eukaryotic cells. Prokaryotic cells have probably been around for 3.5 billion years, while eukaryotic cells arose only about 1 billion years ago. It is thought that eukaryotic cell organelles like nuclei, mitochondria and chloroplasts are derived from prokaryotic cells that became incorporated inside larger prokaryotic cells. This idea is called endosymbiosis, and is supported by these observations: organelles contain circular DNA, like bacteria cells. contain 70S ribosomes, like bacteria cells. organelles have double membranes, as though a single-membrane cell had been engulfed and surrounded by a larger cell. organelles reproduce by binary fission, like bacteria. organelles are very like some bacteria that are alive today.

28 Organic compounds of cell including four classes of molecules :- 1.carbohydrates. 2.lipids 3.proteins 4.nucleic acids. Carbohydrates  The basic formula of these molecules is (CH 2 O) n.  Carbohydrates include mono saccharides (one sugar), disaccharides (two sugars joined together), and poly saccharides (many sugars) joined together.

29 A- Mono saccharides  Simple sugars with carbon backbone of three to seven carbon atoms.  The best known sugars are those that six carbons (hexoses) e.g. glucose, fructose. Have formula (C 6 H 12 O 6 ).  Sugars have five carbons atoms called (pentoses) e.g. Ribose, dexoy ribose found in nucleic acid.  sugars are important fuel for many living organisms, their solubility in water enable them to be transported readily in body fluids.  glucose is the blood sugar of humans represent the most important source of energy.

30 Di saccharide :-  contains two mono saccharide that have joinied by dehydration reaction.  their sugars linked by a glycosidic bond, e. g. Lactose contains galactose and glucose (found in milk) ; Maltose composed of two glucose molecules (is found in our digestive tract as a result of starch digestion) ; and sucrose contain glucose and fructose. Poly saccharides :- Are Made up of three or more mono saccharide linked together.

31 e. g.  Glycogen and starch : are the storage forms of poly saccharides in animal and plants cells.  Chitin and cellulose :- are the structural forms of carbohydrates in animal and plant cell. Lipids  many of these are insoluble in water because lack any polar groups.

32 There are four groups of lipids :- A.fat and oils :- Fats and oils provide  The most concentrated energy reserve available to the organism.  They contain two types of unit molecules fatty acid and glycerol.  Some fatty acids have one or more double bonds between the carbon atoms ((16 or 18 carbon atoms with a carboxyl group (COO-) at one end)) which are described as unsaturated fatty acids.  Fats have mostly saturated fatty acid (so they are solid at room temperature ).

33  Oils tent to have unsaturated fatty acids (they are liquid at room temperature).  Therefore, A diet rich in animal fats (saturated) is linked to the development of diseases of the heart and major arteries.  While unsaturated fats (plant fats or oil) not to have this effect. C – C – C – C – C – | | HO O H H Saturated fatty acids Unsaturated fatty acids C – C – C – C – C – | | | H H H HO O

34 Phospholipids :  They are component of cellular membranes including the plasma membrane it consist of two fatty acids joined to polar head group. Tail hydrophobic Head hydrophilic  In the presence of water, phospholipids head molecule is polarized and the tails are not.

35 Steroid  The most abundant steroid in the human is cholesterol.  Cholesterol is the precursor of several other steroids, including our hormones such as aldosterone, which helps regulate the sodium content of the blood and the sex hormones, which help maintain male and female characteristics. Waxes :-  Waxes are solid at normal temperature and it contain Long chain fatty acids bonds with a long chain alcohol

36  In human wax is produced by glands in the outer ear canal.  Its function is to trap dust and dirt particles preventing them from reaching to the ear drum. Comment :- Lipids have three major roles in cells :- A.They provide an important form of energy storage. B.Lipids, are the major components of cell membrane. C.Play important roles in cell signaling, both as steroid hormones e.g. (estrogen and testosterone) and as messenger molecules that convey signals from cell surface receptors.

37 Proteins 1.Proteins are polymers of 20 different amino acid. 2.Amino acid are very large molecules with structural and metabolic functions. 3.All amino acids contain two important functional groups : 1.A carboxyl (acid) group (-COOH). 2.An amino group (-NH 2 ). Both of which ionize at normal body pH, therefore, amino acid are hydrophilic : C COOHH2NH2N R H Non ionized C COO-H3N+H3N+ R H Ionized

38 Two amino acids are joined by a condensation reaction between the carboxyl group of one and the amino group of another. NCCNCC H H H R O H H R OH O + H 2 O Proteins have structural and metabolic functions : 1.Serve as building and structural elements include :  Actine and Myosin of muscle and other contractile systems.  Collagens which form connective ligaments with the body.  Kertaines which form protective converings such as skin, hairs and nails.

39 2.Proteins that regulate the numerous processes and activities of the organism include :  Enzymes, which modulate chemical reaction of metabolism.  Antibodies, which provide immunity against infection.  Hormones, e.g., insulin that regulate the glucose content of the blood. 3.Proteins are responsible for transporting many materials through the circulatory system. 4. Proteins also serve as food reserves e.g. :  Ovalbumin, which is the chief ingredient of egg white.  Casein, the major protein in milk.

40 Enzymes Enzymes are complex, organic colloidal, catalysts produced by living cells. All enzymes are proteins. Some enzymes consist solely of protein for example: pepsin, trypsin and amylase. Others have a non protein part which is essential to their structure known as a prosthetic group. For example, the respiratory enzyme cytochrome oxidase has an iron containing prosthetic group Other enzymes although are consisting only of protein require the presence of a non protein enzyme cofactor before they can function. Enzymes act as catalysts. that is they speed up the rate of chemical reaction

41 Hormones Hormones are organic chemicals produced by one set of cells that affect different set, or chemicals produced in one part of the body that controls the activity of other parts. Hormones are chains of peptides that are coded for by genes and are synthesized within the cytoplasim at the ribosome, and package into vesicles at the golgi apparatus and secreted at the plasmamembrane. There are several types of animal hormones for example:

42 1 - Insulin: a hormone secreted by the pancreas that lowers the blood glucose level. 2 – Parathyroid hormone (PTH) secreted by the four parathyroid gland that increases the blood calcium level and decreases the blood phosphate level. 3 – Thyroxin (T4) secreted from thyroid gland that promotes growth and development in vertebrates. 4 – Testosterone which is the male sex hormone produce by testes. 5 – The ovaries produced (estrogen and progesterone) which are the female sex hormones. The hypothalamus and pituitary gland control the hormonal secretion of testes and ovaries in the same manner that was described for the thyroid gland previously.

43 6 – Growth Hormone (GH) or somatotropic hormone: If too little GH is produced during childhood the individual comes a pituitary dwarf and if too much is produced, the individual is a pituitary giant. In both instances the individual has normal body proportion. 7 - Prolaction (PRL): produced in quantity only after child birth. In causes the mammary glands in the breasts to develop and produce milk. It also plays a role in carbohydrate and fat metabolism. 8 - Melanocyte – stimulating hormone (MSH): It was stimulated melanocytes to increase their synthesis of melanin.

44 Proteins have four levels of structures : 1.Primary structure : Is the sequence of the amino acids joined by peptide bonds. 2.Secondary structure :  Is the regular arrangement of amino acids within localized regions of poly peptides.  There are two types of it : α he lix and β sheet.  Secondary structures are held together by hydrogen bonds between the CO and NH groups of peptide bonds.

45 3.Tertiary structure : Is the folding and twisting of the polypeptide chains, that is maintained by various types of bonding between the R groups. 4.Quaternary level of structure : It consists of the iteractions between different polypeptide chains in proteins composed of more than one polypeptide. Hemoglobin chains, e.g., is composed of four polypeptide chains.


47 A\Tow major kinds of nucleic acid are found in living things deoxyribonucleic acid or DNA and ribonucleic acid or RNA. B\Each o these is a linear, unbranched polymer. C\The monomers is nucleic acids are nucleoties which composed of :- 1. Five carbon sugar ( pentose ) : ribose (RNA) deoxyribose (DNA) 2. Nitrogen bases (purine, pyrimidine ). DNA contains tow purines (adenine, guanine ) and tow pyrimidines (cytosine and thymine ). D\Adenine, guanine and cytosine are also present in the RNA, but RNA contains Uracil instead of thymine.

48 3. One, tow or three phosphate groups. DNA is doubled strander molecule consisting of two polynucleotide chains running in opposite direction. There is a relationship, called complementarity between bases forming a rung, if one base of pair adenine, the other must be thymine, if one base is guanine the other must be cytosine (figure 3.18). Watson and Crick deduced this structure in 1953. DNA has a unique role as genetic material. DNA

49 It transmits the genetic massage from generation to generation and from cell to cell in an individual. In eukaryotic cells DNA is located in the nucleus. * RNA is usually occurs in single stranded from different types of RNA participate in a number of cellular activities:- 1. Messenger RNA ( mRNA ) : carries information from DNA to the ribosome, where it serves as a template for protein synthesis. 2. Tow other types of RNA ( 2 ribosome RNA (rRNA), and 3 transfer RNA (tRNA)) are involved in protein synthesis. 2. Tow other types of RNA ( 2 ribosome RNA (rRNA), and 3 transfer RNA (tRNA)) are involved in protein synthesis. * RNA found in both the nucleus and cytoplasm * RNA found in both the nucleus and cytoplasm RNA

50 ATP (Adenosine triphosphate) ATP is a nucleotide in which adenosine is composed of adenine and ribose three phosphate groups attached to ribose (the pentose group). ATP with unstable phosphate bonds, is the energy currecy of cells. (hydrolysis) ATP ADP + P + energy H2OH2O

51  Normal metabolic activity depends on very small amount of more than a dozen organic substances called Vitamins.  A vitamin is the general term for a number of chemically unrelated, organic substances that occur in may foods in small amounts and are necessary for the normal metabolic functioning (figure 2002).  Vitamins may be water soluble (B,C), or fat soluble (A,D,E and K). Vitamins :

52 Tools of cell biology A.The compound microscope (CM). B.Electron Microscope (EM). C.Cell Fractionation. D.Growth of animal cells in culture. E.Histochemistry and cytochemisty. F.Auto radiography. G.Viruses.

53 A.The compound Microscope (CM) This lab. Aims to :- 1.Identify and state the function of the primary part of a compound Microscope. 2.Learning how to : (a) carry and used a microscope, (b) focus a microscope, (c) prepare a wet mount, (d) determine the magnification of field of view.

54 Introduction : The light M. remains a basic tool of cell biologists with technical improvements allowing the visualization of ever increasing details of cell structure. There are several different types of light M. are routinely used to study various aspect of cell structure. 1.Simple light M. (single lensed M.) magnified up to 300X (times) their actual size. 2.Compound light microscope (the subject of this lab.) magnified up to 1000X (times). 3.The bright field microscope. 4.Phase contrast microscope.

55 5.Differential interference contrast microscope. 6.Video-enhanced differential interference contrast microscope. 7.Fluorescence microscope. 8.Confocal microscope.


57 Introduction : Because of the limited resolution of the light microscope, analysis of the details of the cell structure has required the use of more powerful microscope techniques, namely Electron Microscope, which was developed in 1930 and first applied to biological specimens by Albert claude, Keith porter, and George palade in the 1940 and 1950. B.Electron Microscope (EM) This Lab. Aims to : to identify the EM and how to use it.

58 There are two types of (EM) : 1.Transmission electron Microscope (TEM) : 2.Scanning Electron Microscope (SEM): An electron microscope that form an image by passing an electron beam through a specimen and focusing the scattered electron with magnetic lenses. An electron microscope that scan a beam of electron over the surface of a specimen and form an image of the surface from the electrons. the most medical investigations are used (TEM) more then (SEM).

59 Tecnia Electron Microscope (1)= Electron gun

60 Tecnia Electron Microscope (2)= The upper part of the column containing the condenser lenses

61 Tecnia Electron Microscope (3)= The specimen area with CompuStage

62 Tecnia Electron Microscope (4)= The lower part of the column with the image-forming lenses

63 Tecnia Electron Microscope (5)= The lower part of the column with the image-forming lenses and the projection chamber

64 Tecnia Electron Microscope (6)= The microscope controls are situated on two movable control panels

65 Tecnia Electron Microscope (7)= Detector systems which are embedded into one single system, with one or two monitor(s), one keyboard and one mouse

66 Tecnia Electron Microscope (8)= The X-ray detector for Energy Dispersive Spectroscopy (EDS) is at the specimen area and its cooling tank can be seen

67 Tecnia Electron Microscope (9)= Wide angle CCD camera or High-Angle Annular Dark Field (HAADF) detector for STEM is located just above the viewing chamber at the ‘35 mm port’

68 Tecnia Electron Microscope (10)= Bright Field (BF) and Dark Field (DF) detectors for STEM are located underneath the viewing screen

69 Principle: The electron microscope can achieve a much greater resolution than that obtained with light microscope, because the wave length of electron is shorter than of light. Thus, under optimal conditions, the resolving power of the electron microscope is approximately 0.2 nm. (Resolution is the ability of microscope to separate or distinguish between small object that are closed together). TEM in principle is similar to the observation of stained cells with the light microscope. Specimens are fixed and stained with salt of heavy metals which provide contrast by scattering electrons.

70 Systems of TEM 1. Illumination systems (High Voltage) A- V- shape tungsten filament: is heated about 2100 °C. The filament is housed in a metal cathode shield (gun cap) having a 1-2 mm. Hole concentrated at the base near the filament tip. This whole unit that accelerates the electrons down to the column is called the electron gun. B- Condenser lens: is electron magnetic lens, used to four the electron beam into the specimen. C- A mechanical stage: It is lies with in the specimen chamber allows lateral movement of the specimen just as with the light microscope

71 2. Imaging systems (Lenses) The most critical lens in the image-forming objective electromagnetic lens just below the specimen, two or more lenses below this magnify the image and project it on the fluorescent screen. 3. The pumping systems : EM have two stage rotary pump setup separately together with its driving motor, and the oil diffusion pump, provide for pumping down and for vacuum measurement.

72 Outline of typical preparation: 1 – Tissue removal: As quickly as possible or practical. 2 – Fixation: Immediate tissue sliced or diced in fixative – 2% glutaraldehyde PH=7.4 for 2h. 3 – Wash: Excee fixative removed by rinse in balanced salt solution such as (Na-cacodylate buffer) for (15) min (3 changes) in 4°C. 4 – Post fixation: With 1% Osmium tetra oxide (OsO4) for 2h. (Continuouo rotation) in 20°C. 5 – Wash: as number 3.

73 Outline of typical preparation: 6 – Dehydration: water removed by graded series of acetone such as follow: 25% acetone for 15 min in 4°C. 50% acetone for 30 min in 4°C. 75% acetone for over night in 4°C. 90% acetone for 30min in 4°C. 100% acetone for 30min (2 changes) in 20°C. 7 – Infiltration: Propylene oxide (Ivol.) + Durcupan mix. 1 – (vol.) 2hr (continuous rotation) in 20°C. Durcupan mix.1 for over night in 20°C. Durcupan mix.2 for 2h. (continuous rotation) in 20°C.

74 Outline of typical preparation: 8 – Embedding: used Durcupan mix.2 (in Beam capsules). 9 – Polymerization: in oven 65°C for 48h. 10 – Block trimming: Tissue exposed at tip of capsule by removal of excess plastic. 11 – Sectioning: Trimmed block with triangular glass knife oriented in ultra microtome to slice sections less than 0.1 µm. (usual 500-800 A°). Sections must be floated off on a water surface. 12 – Mounting: Sections set on fine mesh copper "grids" 3mm in diameter.

75 Outline of typical preparation: 12 – Mounting: Sections set on fine mesh copper "grids" 3mm in diameter. 13 – Staining: Grids placed up side down on a drop of saturated uranyl- Acetate, (15 min). Rinse in distilled water; repeat with lead citrate and rinse. 14 – Examination: with TEM. 15 – Viewing-Photographic systems: Pictures can be taken by advanced types of cameras with high resolution. Pictures can be with two or three dimension. The pictures are then saved on the hard disk of computer or CD's or printed by high resolution printers.

76 C. Cell Fractionation The first step in subcelluar fractionation is the disruption of the plasma membrane, under conditions that do not destroy the internal components of the cell, several methods are used including :- 1.Sonication (exposure to high frequency sound). 2.Grinding in a mechanical homogenizer. 3.Treatment with a high-speed blender. This means separating different parts and organelles of a cell, so that they can be studied in detail. All the processes of cell metabolism (such as respiration or photosynthesis) have been studied in this way. The most common method of fractionating cells is to use differential centrifugation:

77 1. Cut tissue (e.g. liver, heart, leaf, etc) in ice-cold isotonic buffer. Cold to stop enzyme reactions, isotonic to stop osmosis, and buffer to stop pH changes. 2. Grind tissue in a blender to break open cells. 3. Filter. This removes insoluble tissue (e.g. fat, connective tissue, plant cell walls, etc). This filtrate is not called a cell-free extract, and is capable of carrying out most of the normal cell reactions.

78 4. Centrifuge filtrate at low speed (1 000 x g for 10 min) 5. Centrifuge supernatant at medium speed (10 000 x g for 30 min) 6. Centrifuge supernatant at high speed (100 000 x g for 1 hour) 7. Centrifuge supernatant at very high speed (300 000 x g for 3 hours) 8. Supernatant is now organelle-free cytoplasm

79 A more sophisticated separation can be performed by density gradient centrifugation. In this, the cell-free extract is centrifuged in a dense solution (such as sucrose or caesium chloride). The fractions don't pellet, but instead separate out into layers with the densest fractions near the bottom of the tube. The desired layer can then be pipetted off. This is the technique used in the Meselson-Stahl experiment (module 2) and it is also used to separate the two types of ribosomes. The terms 70S and 80S refer to their positions in a density gradient


81 D. D.Growth of animal cells in culture :-  Animal cell culture are initiated by the dispersion of a piece of tissue into suspension of its components cells, which is then added to a culture dish containing nutrient media.  Bacteria and Most animal cell types attach and grow on the plastic surface of dishes used for cell culture.  In addition to slats and glucose, the media used for animal cell cultures contain various amino acids and vitamins, which the cells can not make for themselves.  The growth media for most animal cells in culture also include serum, which serves as a source of polypeptide growth factors that are required to stimulate cell division.

82 E.Histochemistry and cytochemistry :-  This terms are used mainly to indicate methods for localizing different substances, lipids, proteins, carbohydrates, ions, in tissue sections.  Several procedures are used to obtain this type of information, most of them based on specific chemical reactions between macromolecules, both methods usually produced in soluble colored or electron dense compounds that enable the localization of specific substances by means of light or electron microscopy.

83  Several ions (e.g. irons and phosphate) have been localized in tissue with these methods. F.Auto radiography :-  Cells are incubated with molecules that will be specifically in corporated into certain cell constituent.  These molecules are radioactively labeled that are they contain one or more several atoms that are radioactive isotopes. e.g. thymines is found in the DNA but not in RNA, so incorporated of thymine in to cells indicates specifically that DNA synthesis is occurring.

84 G.Viruses :- Are the smallest infectious agents (20-300 nm in diameter), containing as their genome a molecule of either RNA or DNA. All viruses are parasites and which replicates within living cells. This can be a plant, animal or bacterium, most viruses cause disease. Most of our knowledge of viruses come from work on (bacteriophages). These are viruses that infect only bacteria.

85 How do viruses cause disease? When a virus enters a cell in your body, it takes over control of it the cell is made to stop all its normal work and start making more viruses. Eventually, the cell bursts releasing the new viruses. Each of these new viruses will enter another cell and reproduce, in few hours, tens of thousands of cells can be destroyed. This destruction of cells usually results in disease. Viral diseases can often be very serious, because there are no drugs to fight them. lt is up to the body to fight them be making (antibodies). Viruses lack almost everything normally found in an ordinary cell.

86 They have no ribosomes, nor any of the enzymes necessary for protein synthesis, and have none of the enzymes needed for energy production, they usually contain only those enzymes necessary for them to invade a cell and replicate their own genetic material (DNA or RNA) virus particles are either polyhedral or helical or a combination of both. Viruses show two main types of the life history 1- lytic cycle, 2- lysogenic cycle.

87 Bacteriophages A bacteriophage is a virus which multiplies within living cells, in this case bacteria. It is probable that every type of known bacterium is host to at least one phage, although any given phage is host-specific. Bacteriophages have peculiar structure consist of a head & a tail the head contains nucleic acid surrounded by a protein membrane. They contain more than 40% of DNA. Bacteriophage attack a bacterium it penetrates the membrane with its tail and then proceeds to inject nucleic acid (DNA or RNA) from its head into the bacterium. Multiplication takes place until the bacterium becomes filled with phages which develop & finally are released by the brushing of the membrane.


89 The phage are of 2 types:- 1 ) Virulent (cause lytic cycle) After penetraining & injecting the nucleic acid and passing the eclipse phase (virus through which is visible). Then maturation phase and assembling & developing tails (multiplication). The viruses will rupture bacterial wall in order to release. 2 ) Temperate (non virulent cause lysogenic cycle) Phage of this type do not destroy the bacteria but passes the same steps of the virulent type except without destroying the bacteria. Temperate bacteriophage change into virulent by using ultra violet rays.

90 Viruses and cancer: Cancer is a family of disease characterized by.uncontrolled cell proliferation. The growth of normal animal cells is carefully regulated to meet the needs of the complete organism. In contrast, cancer cells grow in an unregulated manner, ultimately invading and interfering with the function of normal tissues and organs. The human cancers that are caused by viruses include cervical and other anogenital cancers (papillomaviruses) liver cancer, (hepatitis B virus), and some types of lymphomas (Epstein-Barr virus and human 1-cell lymphotropic virus).

91 Retroviruse is a type of RNA viruses cause human immunodeficiency viruses (HIV) or called (AIDS) and also cause certain forms of cancer.

92 Bacteria and food poisoning Some of the bacteria that can live on our food, can also cause disease. They do this by :- 1 ) Excreting toxins into the food which will poison our body, when eating this food. 2 ) Infecting the body after being eaten with the food the food poisoning is restricted to infection by enteric pathogens contaminating food, or ingestion of food containing exotoxins. These can range from sickness and diarrhea to severe intestinal bleeding and even death. For example, salmonella spp (Typhoid & food poisoning). Brucella melitensis (Malta fever).

93 Metric units of linear measurement SizeAbbreviationUnit 39.37 U.S inches mMeter 10-2 meter cmCentimeter 10-3 meter mmMillimeter 10-6 meter umMicrometer 10-9 meter nmNanometer 10-10 meter AAngstrom

94 Dietary Carbohydrate and disease : This is of profound public health importance because o the clearly defined Negative effect of obesity especially when centrally distributed in relation to diabetes, Coronary heart disease and other chronic disease of life style. Genetic and environ mental factors play a role In deter mining the propen sity for obesity. High carbohydrate food and and lack of phsical activity promote to the in creasing rates of obesity.

95 Foods containing sugars or starch may be easily broken down by a-amylase and bacteria in the mouth and can produce acid which increases the risk of carrier. With in all populations a family history of NIDDM is an important predis posing factory. Diet and life style-relation conditions which may lead to obesity will cearly in flounce the risk of developing NIDDM.

96 Foods rich in non-starch poly sacch aridy and Carbohydrate-containing foods with alow glycemic- index appear to protect against diabetes. Many genetic and life style factors are in volved in the etiology of Coronary heart. Dietary factors may in fluence these processes direclty or Via arange of Cardio vascular disease. Dietary factors may in fluence these processes direclty or Via arange of Cardio vascular disease.Obesity. High in takes of some saturated fatty acid, increasing carbohydrate in take

97 Obesity is an important risk bifido bacteria and lacto bacilli in the gut and thus reduce the risk of ucut infective gastro in testinal ill nesses.

98 How are lipid storage disease inherited Lipid storage disease are inherited from one or both parents who carry a defective gene that regulates a particular protein in a class of the body's cells. They can be inherited two ways:- a. Autosomal rescessive inheritance occurs when both parents carry pass on a copy of the faulty gene, but neither parent is affected by the disorder. b. X-linked (or sex-linked) recessive inheritance occurs when the mother carries the affected gene on the X chromosome that determine the child's gender passes in to her son.

99 How are these disorder diagnosed Diagnosis is made through clinical examination, biopsy, genetic testing, mole cular analysis of cells or tissue to dentify inherited metabolic disorder and enzyme assays (testing a variety of cells or body fluids in culture for enzyme deficiency).

100 1 – Gaucher disease:- is the most common of the lipid storage diseases. It's caused by a deficiency of the enzyme glucocerebrosidase. Fatty material can collect in the spleen, Liver, Kidney, Lungs, brain and bone marrow.

101 2 – Niemann-pick disease Disease is actually a group of outosomal recessive disorders caused by an accumulation of a fat and cholesterol in cells of the liver, spleen, bone marrowm lunge and in some patients brain.

102 Alpha-galacto sidase-A deficiency cause a build up of fatty material in the autonomic nervous system, eyes, kidneys, and cardio vascular system. Alpha-galacto sidase-A deficiency cause a build up of fatty material in the autonomic nervous system, eyes, kidneys, and cardio vascular system.

103 Also know as Farber’s lipogranulomatosis or ceramidase deficiency. Cause an accumulation of fatty material in the toints tissues, and central nervous system.

104 Also known as GM 2 variant B. Caused by adeficieny in the en zyme beta- hexosaminidase A. Caused by adeficieny in the en zyme beta- hexosaminidase A. Affected children appear to develop normally for first few months of life. Affected children appear to develop normally for first few months of life.

105 This is a a sever form of Tay-sachs disease occurs at the age 6 months. Gala etosylceramide lipidosis caused by deficiency of the enzyme galac to sylceramidase.

106 Plasma Membrane " Structure and Function "  The structure and function of cells are critically dependent on membranes.  Universally, a plasma membrane protects a cell by acting as. A barrier between its Living Contents and surrounding environment.  It regulates what goes into and out of the cell and markes the cell as being unique to the organism.  In multi cellular organisms, cell junctions requiring specialized features of the plasma membranes connect cells together in specific ways and pass on information to neighboring cells so that the activities of tissues and organs are coordinated.

107  Investigators noted that Lipid – Soluble molecules entered cells more rapidly than water – soluble molecules.  This promoted them to suggest that Lipid are a component of the plasma membrane.  The formation of biological membranes is based on the properties of lipids, and all cell membranes share a common structural organization, Bilayers of phospholipids with associated proteins.

108 Membrane Lipids  Lipid constitute 50% of the mass of most cell membranes, although this proportion varies depending on the type of membrane :- Phospholipids :-  The fundamental building blocks of all cell membranes, which are amphipathic molecules, consisting of two hydrophobic fatty acid chains linked to a phosphate containing hydrophilic head group.  The hydrophilic (polar) heads of the phospholipids molecules face the intercellular and extra cellular fluids.

109  The hydrophobic (non polar) tail face each other in the membrane interior. Glycolipids :-  have a structure similar to phospholipids except that the hydrophilic head is a variety of sugars Joined to form a straight or branching Carbohydrate chain.  Glycolipids have a protective function. Cholesterol :-  is a lipid that is found in animal plasma membranes.

110  cholesterol reduces the permeability of the membrane to the most biological molecules. Membrane Proteins  Proteins constituting 25 to 75% of the mass the various membranes of the cells.  Membrane proteins carry out the specific functions of the different membranes of the cell.  These proteins are divided into two general classes, based on the nature of their association with the membrane.

111 1.Integral membrane proteins, are embedded directly within the lipid bilayer.  Many integral membrane proteins called ((transmembrane proteins)) span the lipid bilayer with portions exposed on both sides of the membrane. 2.Peripheral membrane proteins are not inserted into the lipid bilayer but are associated with the membrane indirectly, generally by interactions with integral membrane proteins.  The carbohydrate chains of glycolipids and glycoproteins serves as the fingerprints of the cell.

112  The lipid and protein composition of the inside half differs from the outside half.  The carbohydrate chains of the glycolipids and glycoproteins form a carbohydrate coat that envelops the outer surface of the plasma membrane.  On the side some proteins serve as links to the cyto skeletal filaments and on the out side some serve as links to extra cellular matrix.

113 Membrane Protein Diversity These are some of functions performed by proteins found in the plasma membrane. A.Channel Protein :-  Allows a particular molecule or ion to cross the plasma membrane freely.  Cystic fibrosis an inherited disorder is caused by faulty chloride (Cl-) channel ; a thick mucus collects in air ways and in pancreatic and liver ducts.

114 B.Carrier protein :  Selectively interacts with a specific molecule or ion so that it can cross the plasma membrane.  The inability of some persons to use energy to sodium- potassium (Na+ - K+) transport has been suggested as the cause of their obesity. C.Cell recognition protein :  Major histo compatibility complex (MHC) glycoproteins are different for each persons, so organ transplants are difficult to achieve.

115  Cells with foreign MHC glycoproteins are attacked by blood cells responsible for immunity. D.Receptor Protein  is shaped in such a way that a specific molecule can bind to it.  Pygmies are short, not because they do not produce enough growth hormone, but because their plasma membrane growth hormone receptors are faulty and cannot interact with growth hormone.

116 E.Enzymatic Protein  Catalyzes a specific reaction.  The membrane protein, adenylate cyclase is involved in ATP metabolism, Cholera, Bacteria release a toxin that interferes with the proper functioning of adenylate cyclase ; sodium ions and water leave in testinal cells and individual dies from severe diarrhea.

117 How molecules cross the plasma membrance How molecules cross the plasma membrance  The plasma membrane is semi permeable, allows some molecules to pass through (e.g. : small, non charged, lipid – soluble molecules).  Plasma membrane also is often regarded as differentially permeable (or selectively permeable), because not all small molecules can freely pass through it.

118 Molecules cross the plasma membrane in two ways :-

119 B.Active ways use energy: Involve; active transport, endocytosis and exocytosis. Passive Ways A.Diffusion :- occurs when molecules move from higher to lower concentration – that is, down their concentration gradient, until they are distributed equally, e.g. when a few crystals of dye (solute) are placed in water (solvent). A.Passive ways which do not use energy. involve: diffusion and facilitated transport.

120  Gases can also diffuse through the lipid bi layer, this is the mechanism by which oxygen enters cells and carbon dioxide exits cells.  As an example, consider the movement of oxygen from the air sacs (alveoli) of lungs to blood in the lung capillaries. Osmosis :-  The diffusion of water across a differentially permeable membrane has been given a special name it is called Osmosis.  Osmotic pressure develops on the side of the membrane that has the higher solute concentration.

121  A thistle tube convered at the broad end by a permeable membrane contains a 10% sugar solution and is them placed in a beaker containing a 5% sugar solution.  Water molecules pass through membrane more readily than do sugar molecules.

122 Tonicity :-  Tonicity refers to the strength of a solution in relation ship to osmosis.  Cells can be placed in solutions that have the same percentage of solute (isotonic solution) a higher percentage of solute (hypertonic solution). or a lower percentage of solute (hypotonic solution), than the cell. Isotonic Solution :-  Solutions that cause cells neither to gain nor to lose water, that is the solute concentration is the same on both sides of the membrane.

123  A 0.9% solution of the salt sodium chloride (NaCl) is known to be Isotonic to red blood cells because the cells neither swell nor shrink where placed in this solution.  Therefore, physician must put this point in his mind when giving blood or fluid to the patients. Hypertonic Solution :-  Solutions that cause cells to shrink or to shrivel due to loss of water.  Any concentration with a concentration higher than 0.9% Sodium chloride is hypertonic to red blood cells

124 Hypotonic Solution :-  Solutions that cause cells to swell or even to burst, due to an in take of water.  Any concentration of salt solution lower than 0.9% is hypotonic to red blood cell.

125 B.Facilitated transport :-  During facilitated transport, a carrier protein assists the movement of a molecules down it's concentration gradient no energy is required.

126 Active Ways a.Active transport :-  Some molecules and ions can be transported across cell membrane against their concentration gradient if the approper transport enzymes and a source of energy are available.  Proteins involved in active transport often are called pumps, for example, the sodium – potassium pump carries (Na+) to the outside of the cell and (K+) to the inside of the cell.

127 Active transport

128 b.Exocytosis :-  During exocytosis, Vesicles often formed by the Golgi apparatus and carring a specific molecules fuse with the plasma membrane and secretion occurs.  This is the way that insulin leaves insulin – secreting cells,for instance.

129 c.Endocytosis  During endocytosis, cells take in substances by vesicle formation.  A protein of the plasma membrane in vaginates to envelop the substance, and then the membrane pinches off to from an intracellular vesicle. There are Three Methods of Endocytosis 1.Phagocytosis :- means "cell eating", occurs when large materials taken inside the cell, such as a food particle or another cell.

130  White blood cells can engulf bacteria and worn- out red blood cells by phgocytosis Digestion occurs when the resulting vacuole fuses with a lysosome. 2.Pinocytosis :- means "cell drinking", occurs when vesicles from a round a liquid or very small particles.  Blood cells,and that line the kidney tubules or in testinal wall, use this method of ingesting substances. 3.Receptor – mediated endocytosis :- is a form of pinocytosis that is quite specific because it involves the se of a receptor protein shaped in such a way that a specific molecule can bind to it.

131  A macromolecule that binds to a plasma membrane receptor is called a ligand.

132 Cell Membrane Specialization The lateral parts of the cell membrane can show, several specialization that form "intercellular junctions" functions of these junctions :- 1.They are the sites of adhesion between adjacent cell. 2.They prevent the flow of materials through the intercellular. 3.They help in the intercellular communication.

133 There are Three Types of Junctions 1.Adhesion Junctions (desmosomes) :-  In this type, the internal cytoplasmic plaques firmly attached to the cytoskeleton with in each cell are joined by intercellular filaments.  In some organs like the heart, stomach, bladder, adhesion junctions hold the cell together. 2.Tight junctions :  Adjacent cells are even more closely joined by tight junctions in which plasma membrane proteins actually attach to each other producing a zipper like fastening.

134  These junctions between cells from an impermeable barrier and prevent the flow of materials in intercellular space. e.g. in the kidneys the urine stays within kidney tubules because the cells are joined by tight junctions.

135 3.Gap junction :-  It allows cells to communicate, and is formed when two in identical plasma membrane channels join.  The channel of each cell is lined by six plasma membrane proteins (Hexamers).

136 Functions :- 1.It lends strength to the cells 2.It allows small molecules and ions to pass between them. Gap junctions are important in heart muscle and smooth muscle because they permit a flow of ions that is required for the cells to contract. Apical Modification of Plasma Membrane  Microvilli :  Finger like extensions of plasma membrane that are particularly abundant on the surface of the cells, involved in the absorption. such as the epithelial cells lining the intestine.

137  Stereocilia :  Specialized forms of microvilli.  The stereocilia of auditory hair cells, are responsible for hearing by detecting sound vibrations.

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