Transcript Prokaryotic cells

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 .
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.
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.
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.
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.
Cations:
Calcium ions, Ca2+, 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.
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, Mg2+, 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.
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, PO43-, 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.
Our food consists of chemicals that are
necessary to :1\ Provide energy.
2\ Rerpair tissue.
3\ Regulate body machine.
Water is essential to life :The existence of living things is dependent on
the chemical and physical characteristics of
water :1\ The temperature of liquid water rises and falls
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
systems.
4\ Water has a high surface tension.
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 )
In contrast , non polar molecules , which cannot
interact with water , are poorly soluble in an
aquous environment ( Hydrophobic ).
When water ionizes , it releases an equal number
+
of hydrogen ( H ) and hydrogen ions ( OH ) :H–O–H
H+
+ OH-
Acids and Basses
Acids ( High H+ concentrantion ):Are molecules that dissociate in water ,
releasing hydrogen ions (H+) , so called
proton donor.
For example , an improvtant inorganic acid
is hydrochloric acid ( HCL ) , which
dissociation in this manner :-
HCl
H+ + CL-
Dissociation is almost complete ; therefore , this
is called a strong acid . If hydrochloric acid is
added to a breaker of water , the number of
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
inorganic base is sodium hydroxide ( NaOH ) ,
which dissociates in this manners
NaOH
Na+ + OH-
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.
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- ).
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.
Hydrochloric acid
0.0
Stomach acid
1.0
Normal rain water
5.6
Urine
6.0
Saliva
6.5
Pure water , tears
7.0
Human blood
7.4
Sea water
8.0
Sodium hydroxide
14
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 CH2CO3 is a weak acid that minimally dissociates
and then reforms in the following manner :H2CO3
H+ + HCO3-
♦ Blood always contains some carbonic acid and some
bicarbonate ions .
When hydrogen ions (H+) are added to blood , the following
reaction occurs :H+ + HCO3-
H2CO3
♦ When hydroxide ions (OH-) are added to blood this
reaction occurs :OH- + H2CO3
HCO3- + H2O
These reactions prevent any significant change in blood pH.
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 .
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 .
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) .
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 .
Summary of the Differences Between Prokaryotic and
Eukaryotic Cells
Prokaryotic Cells
Eukaryotic cells
small cells (< 5 mm)
larger cells (> 10 mm)
always unicellular
often multicellular
no nucleus or any membrane-bound
organelles, such as mitochondria
always have nucleus and other
membrane-bound organelles
DNA is circular, without proteins
DNA is linear and associated with proteins
to form chromatin
ribosomes are small (70S)
ribosomes are large (80S)
no cytoskeleton
always has a cytoskeleton
motility by rigid rotating flagellum made
of flagellin
motility by flexible waving undulipodium,
made of tubulin
cell division is by binary fission
cell division is by mitosis or meiosis
reproduction is always asexual
reproduction is asexual or sexual
huge variety of metabolic pathways
common metabolic pathways
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.
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 (CH2O)n .
 Carbohydrates include mono saccharides (one sugar) ,
disaccharides (two sugars joined together), and poly
saccharides (many sugars) joined together.
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 (C6H12O6).
 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 .
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.
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 .
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 ).
 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.
H H H H
| | | |
O
C–C–C–C–C–
| | | |
HO
H H H H
Saturated fatty acids
O
HO
H H H H
| | | |
C–C–C–C–C–
|
|
H
H
Unsaturated fatty acids
Phospholipids :
 They are component of cellular membranes including the
plasma membrane it consist of two fatty acids joined to polar
head group.
Head hydrophilic
Tail hydrophobic
 In the presence of water , phospholipids head molecule is
polarized and the tails are not .
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
 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.
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
groups :
contain two important functional
1. A carboxyl (acid) group (-COOH).
2. An amino group (-NH2).
Both of which ionize at normal body pH , therefore ,
amino acid are hydrophilic :
H
H 2N
C
H
COOH
H 3N +
C
R
R
Non ionized
Ionized
COO-
Two amino acids are joined by a condensation reaction
between the carboxyl group of one and the amino group of
another .
H
H
H
O
N
C
C
R
R
N
H
C
H
OH
+ H2O
C
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 .
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 .
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
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:
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.
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.
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.
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.
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.
3. One , tow or three phosphate groups.
DNA
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.
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
*
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.
* RNA found in both the nucleus and
cytoplasm
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
H2O
ADP + P + energy
Vitamins :
 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).
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 .
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 .
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 .
5. Differential interference contrast microscope .
6. Video-enhanced differential interference contrast microscope .
7. Fluorescence microscope .
8. Confocal microscope .
B.Electron Microscope (EM)
This Lab. Aims to : to identify the EM and how to use it .
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 .
There are two types of (EM) :
1.Transmission electron Microscope (TEM) :
An electron microscope that form an image by passing an
electron beam through a specimen and focusing the
scattered electron with magnetic lenses .
2. Scanning Electron Microscope (SEM):
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) .
(1)= Electron gun
Tecnia Electron Microscope
(2)= The upper part of the
column
containing
the
condenser lenses
Tecnia Electron Microscope
(3)= The specimen area with
CompuStage
Tecnia Electron Microscope
(4)= The lower part of the
column with the image-forming
lenses
Tecnia Electron Microscope
(5)= The lower part of the
column with the image-forming
lenses and the projection
chamber
Tecnia Electron Microscope
(6)= The microscope controls are situated on two
movable control panels
Tecnia Electron Microscope
(7)= Detector systems which are
embedded into one single system, with
one or two monitor(s), one keyboard and
one mouse
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
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’
Tecnia Electron Microscope
(10)= Bright Field (BF) and Dark Field (DF) detectors for STEM
are located underneath the viewing screen
Tecnia Electron Microscope
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.
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
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.
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.
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.
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.
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 uranylAcetate, (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.
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:
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.
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
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
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 .
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 .
 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 .
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.
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.
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.
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.
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.
bacteriophage change into virulent by using ultra violet rays.
Temperate
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).
Retroviruse is a type of RNA viruses cause human
immunodeficiency viruses (HIV) or called (AIDS) and
also cause certain forms of cancer.
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).
Metric units of linear measurement
Unit
Abbreviation
Size
Meter
m
39.37 U.S inches
Centimeter
cm
10-2 meter
Millimeter
mm
10-3 meter
Micrometer
um
10-6 meter
Nanometer
nm
10-9 meter
Angstrom
A
10-10 meter
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.
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.
Foods rich in non-starch poly sacch aridy and
Carbohydrate-containing foods with alow glycemicindex 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.
Obesity.
High in takes of some saturated fatty acid, increasing
carbohydrate in take
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.
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.
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).
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.
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.
Alpha-galacto sidase-A deficiency cause a build
up of fatty material in the autonomic nervous
system, eyes, kidneys, and cardio vascular
system.
Also know as Farber’s lipogranulomatosis or
ceramidase deficiency.
Cause an accumulation of fatty material in the
toints tissues , and central nervous system.
Also known as GM2 variant B.
Caused by adeficieny in the en zyme betahexosaminidase A .
Affected children appear to develop normally for first
few months of life.
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.
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 .
 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
associated proteins .
,
Bilayers
of
phospholipids
with
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 .
 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.
 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 .
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 .
 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 .
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 .
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 sodiumpotassium (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 .
 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 .
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 .
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 .
Molecules cross the plasma membrane in two ways :-
A. Passive ways which do not use energy. involve: diffusion and facilitated
transport .
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) .
 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 .
 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 .
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.
 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
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 .
B. Facilitated transport : During facilitated transport , a carrier protein assists the
movement of a molecules down it's concentration gradient
no energy is required .
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 .
Active transport
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 .
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 .
 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 .
 A macromolecule that binds to a plasma membrane receptor
is called a ligand .
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 .
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 .
 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 .
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) .
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.
 Stereocilia :
 Specialized forms of microvilli .
 The stereocilia of auditory hair cells , are responsible for
hearing by detecting sound vibrations .