BIOLOGY

Topic 2
CELLS, CELL TRANSPORT and
IMMUNITY.

Cells
The cell is the basic unit of living organisms. Organisms can be divided into two main
groups, prokaryotes and eukaryotes according to their cell structure.

Bacteria are prokaryotes and are very small single celled organisms with no nucleus and
no membrane-bound organelles in their cytoplasm.

A typical prokaryotic cell will always contain:

 ∙ a cell wall (made of murein – a glycoprotein)
∙ a cell surface membrane
∙ a circular DNA molecule that is free in the cytoplasm and not associated with protein
∙ ribosomes (smaller than in eukaryotes)
∙ cytoplasm.

Structures that may be present in a prokaryotic cell include:

∙ a capsule surrounding the cell wall

∙ one or more plasmids
∙ one or more flagella.

Eukaryotes

∙ Eukaryotes form the vast majority of living organisms including plant and animal
cells.

∙ Eukaryotes are characterised by the presence of a nucleus and a cytoplasm

containing many cell organelles e.g. mitochondria and ribosomes.

∙ The diagram below shows a typical eukaryotic animal cell.

Animal cell
 Comparison of Prokaryotes and Eukaryotes
Prokaryotic cell Eukaryotic cell

1 No nucleus present Nucleus present

2 No mitochondria present Mitochondria present

3 No Golgi body Golgi body present

4 No endoplasmic reticulum Endoplasmic reticulum present

5 Smaller, 70S ribosomes Larger, 80S ribosomes

6 DNA is circular and not DNA is linear and associated

associated with proteins with proteins

7. Cell wall contains murein Cell wall (if present) does not
contain murein

Eukaryote plant cell

Plant cells also have additional structures not present in animal cells.

∙ A cell wall providing support, strength and shape to the cell. The cell wall of plants*
consist of cellulose microfibrils (which provide strength) embedded in a matrix
containing other polysaccharides such as hemicelluloses.

∙ Chloroplasts, (in photosynthetic cells), containing the pigment chlorophyll.

∙ A large vacuole containing soluble sugars, salts and sometimes pigment. The membrane
surrounding the vacuole is known as the tonoplast.

NOTE

1. The cell wall of algae consists of either cellulose or glycoproteins or a mixture of

both.

2. The cell walls of fungi do not contain cellulose but has the nitrogen-containing
polysaccharide, chitin, a polysaccharide called glycan and glycoproteins.
Cell differentiation
As multicellular organisms evolved, specialised cells developed to perform specific
functions. The development of cells into specialised types e.g. neurones, muscle cells, is
known as cell differentiation. These cells are usually grouped together to form tissues,
tissues are organised into organs and organs into systems.

∙ Tissues are are groups of similar cells that perform a specific function and have a
common origin e.g. epithelial tissue, nervous tissue in animals, phloem, xylem tissues
in plants. Blood is classed as a tissue because its cells come from the same type of
stem cell.

∙ An organ is a structure consisting of different tissues, which has a specific

physiological function e.g. the stomach which has a role in the digestion of
food.

∙ Several organs combine to form a system e.g. the stomach, liver and pancreas all
form part of the digestive system.
An epithelial cell from the small intestine

∙ Epithelial cells in the small intestine are specifically adapted for the absorption of
digested food products.

∙ The cell surface membrane is folded into microvilli providing a large surface area for
absorption of digested food (e.g. glucose and amino acids).

∙ Numerous mitochondria are present providing energy in the form of ATP for the active
uptake of digested food molecules.

microvilli
Palisade mesophyll cells

A palisade mesophyll cell from a leaf shows the typical structure of a plant cell. These are
found below the upper epidermis of a leaf and they are adapted for photosynthesis.

∙ They possess numerous chloroplasts to absorb light.

∙ They have thin cell walls providing a short diffusion pathway for entry of carbon
dioxide.

∙ The cells are vertically arranged so there are fewer cell walls for light to pass through.
Viral structure
Viruses are acellular and non-living. They have no nucleus, no organelles, no cell
surface membrane and no cytoplasm.

Viruses are intracellular parasites and can only replicate inside their living host cells
using the cell’s metabolic processes. They cause disease by the combined effects of:

1. Damage to host cells following entry and replication of viruses.

2. Toxins produced in the process of replication.

Viruses

A typical virus particle will always contain genetic material (DNA or RNA), a few
enzymes, a capsid (consisting of protein sub units called capsomeres) and attachment
proteins (e.g. glycoprotein spikes) on the outside.

These are used for attachment or entry into the 'host' cell and also represent the antigens
against which antibodies may be produced.

Some viruses possess an outer lipid envelope which aids the entry of the virus into the
cell by fusing with the plasma membrane (high phospholipid content).
Replication of viruses

∙ The virus particles attach to specific host cells using their attachment
proteins/glycoproteins which are complementary to receptors (often
glycoproteins) on the cell-surface membrane of host cells.

∙ Most viruses inject/insert their nucleic acid (RNA or DNA) into the host cells.

∙ The nucleic acid is used as a set of instructions to direct the replication of more virus
particles using the organelles of the host cell.

∙ This involves producing copies of the viral nucleic acids and proteins (e.g. new
capsids) and other structures e.g. envelopes to form complete viruses which are
often released by lysis of the cell. These virus particles then infect other cells.
Cell organelles

Cell organelles found in eukaryotic cells include the nucleus, mitochondria (singular
mitochondrion), Golgi body, rough and smooth endoplasmic reticulum, chloroplasts and
ribosomes.

Nucleus

The nucleus contains the genetic material, DNA, determining the development, structure
and function of the cell.
The nucleus is bound by a double membrane, the nuclear envelope which has nuclear
pores allowing communication with the cytoplasm

∙ It contains chromatin
(protein bound linear DNA),
and one or more nucleoli
(RNA).

∙ In a dividing cell the

chromatin is in the form of
chromosomes

∙ The nucleus controls protein

synthesis and thus the
development and function of
a cell..
Ribosomes
∙ Ribosomes are very
small
organelles, made up
of protein
and ribosomal RNA
(rRNA).

∙ Ribosomes can be
present in
the cytoplasm singly
or
attached to the RER

∙ They are used in protein

synthesis, joining amino acids
together
Endoplasmic reticulum

Consists of flattened membrane sacs which form an internal transport system in the cell

∙ The surface of the rough

endoplasmic
reticulum (RER) has
ribosomes that
produce secretory proteins i.e.
proteins
exported out of the cell.

∙ Theses secretory proteins are

sent to the
Golgi body for modification
and/or
packaging.

∙ The smooth endoplasmic reticulum

lacks ribosomes and is involved in the
production and transport of lipids

Golgi apparatus

As with the endoplasmic reticulum the Golgi apparatus consists of flattened, membrane
sacs.
It has several functions;

∙ Adds carbohydrates to proteins received from RER to form glycoproteins ∙

Packages proteins/glycoproteins in Golgi vesicles for secretion e.g. enzymes ∙
Produces lysosomes – a type of Golgi vesicle that contains/releases lysozymes
(hydrolytic enzymes).

The Golgi apparatus is abundant in secretory cells e.g. in the pancreas

Golgi
apparatus

Lysosomes

∙ Lysosomes are simple sacs containing digestive (hydrolytic) enzymes (e.g.

lysozyme) surrounded by a single membrane.

∙ The enzymes have to be kept apart from the rest of the cell or they would destroy it.

∙ Are formed by the Golgi apparatus

Functions:

1. Digestion of material taken in by phagocytosis - Lysosomes fuse with the

 vesicles formed during phagocytosis, release their hydrolytic enzymes and digest
(hydrolyse) the material inside.

2. Non-functioning organelles within the cell are engulfed and digested within
lysosomes.

3. Release of enzymes outside the cell – sometimes the enzymes of lysosomes are
released from the cell.
Mitochondria

Mitochondria are involved in aerobic respiration which produces ATP.

They are variable in shape and size, but are often rod shaped.

∙ Mitochondria are bounded by two membranes forming an envelope around an inner

matrix.

∙ Between the outer smooth membrane and the inner folded membrane is the inter
membrane space.

∙ The folds of the inner membrane are called cristae.

∙ The matrix contains enzymes for respiration and also contains circular DNA and
ribosomes.

∙ Cells requiring large amounts of ATP (e.g. involved in active transport) have numerous
mitochondria.
Chloroplasts

∙ Only found in photosynthetic plant cells and algae, varying in number from 1 to100 ∙
Flattened biconvex discs surrounded by an envelope consisting of two membranes

∙ The envelope encloses a membrane system consisting of many flattened sacs called
thylakoids which in places form stacks called grana

∙ These provide a large surface area for the chlorophyll molecules which absorb light for
photosynthesis

∙ The membrane system is surrounded by the stroma which contains enzymes, sugars
and starch granules.

Cell fractionation and ultracentrifugation

Centrifugation separates structures of different density. Differential centrifugation involves

centrifuging at different speeds (forces) and can be used to separate and isolate the
different organelles in a cell.

∙ The cells are first broken open by grinding (homogenising) a tissue such as liver in an
ice cold, isotonic, buffer solution using a blender

∙ An isotonic solution prevents the osmotic movement of water in or out of organelles,

which might cause them to burst or shrivel.

∙ A low temperature (ice cold) prevents the action of enzymes within the cells that might
cause self-digestion (autolysis) of the organelles.

∙ A buffer solution maintains the pH so that proteins particularly enzymes are not
denatured

1. The resulting suspension may be

filtered to remove cell debris such as
cell walls of plant cells or any whole
 Low sediment contains the rest of the
Speed organelles and can be spun at a
higher speed (force) for a longer time
period.

4. This procedure is repeated

increasing the speed and duration of
centrifugation in order to obtain a
series of pellets containing
organelles of decreasing density.

Medium 5. The organelles are usually isolated

Speed in the order; nuclei, chloroplasts (if a
plant cell), mitochondria,

High
Speed

Ribosomes and
soluble proteins
cells that have not burst in
homogenisation.

2. The homogenate is then

centrifuged at low speed. The
endoplasmic reticulum and finally
densest organelles - the nuclei - ribosomes.
form a pellet (sediment) at the
bottom of the tube.

3. The supernatant liquid above the

Microscopy

Light (optical) Microscopy

Light passes through a specimen usually

mounted on a
glass slide. Preparation of slides is usually quick and
easy.

The specimen must:

∙ Be thin, so that light passes through it and so that

a single layer (or few layers) of cells is visible.
∙ Be stained in order to make structures visible.

Light microscopes have relatively poor resolution and so

very small structures are not visible.

Electron Microscopy

The use of the electron microscope has

proved invaluable as a means of
investigating the fine structure
(ultrastructure) of a cell.

The electron microscope uses a beam of

electrons focused by electromagnets as
opposed to the focusing of light rays by
lenses in light microscopy.

Advantages of electron microscopy

∙ Electron microscopy uses electrons that have a shorter wavelength than light and thus
provide greater resolution i.e. the ability to distinguish between two close objects.

∙ At higher magnifications images become blurred when using light microscopy due to poor
resolution. (Note: In exams you should not suggest that higher magnification is an
advantage of using electron microscopes.

The diagram below illustrates the resolution at low and high power of the two different
microscopes.

Disadvantages of electron microscopy

∙ As a vacuum is required living specimens cannot be seen.

∙ Preparation and staining techniques are more complicated than with light microscopy
and can produce artefacts (‘false images arising from poor technique’).

∙ Expensive to buy and expert training is required in their use.

Transmission and scanning electron microscopes

There are two main types of electron microscope – the transmission electron
microscope and the scanning electron microscope.

In a transmission electron microscope (TEM):

∙ A beam of electrons is transmitted through the specimen.

∙ The specimen must be thin and is stained using electron dense substances such as
heavy metal salts

∙ These substances deflect the electrons in the beam and the pattern that the
remaining electrons produce as they pass through the specimen is converted to an
image.

In a scanning electron microscope (SEM):

∙ The specimen is coated with a thin film of a heavy metal such as gold ∙

The electron beam is scanned to and fro across the specimen

∙ Electrons that are reflected from the surface are collected and produce an image on a
viewing screen.

TEM of a mitochondrion SEM of mitochondria

Comparing light (optical) with electron microscopes:
Electron Light

Uses beam of electrons Uses beam of light

Much greater resolution Relatively low resolution

Focused using magnets Focused using glass lenses

Much greater detail / much smaller Much lower detail / smaller structures
structures visible. not visible.
Specimens are dead and dehydrated. Specimens can be living (movement can
be observed).

Image not in colour Image in colour

Preparation of specimen very complex Preparation of specimen relatively easy

and time-consuming. and quick.

Comparing SEM with TEM:

TEM SEM

Higher resolution than SEM Lower resolution than TEM

Produces image of internal structures Produces image of

external/surface structures

Gives a 2D image Gives a 3D image

Sections must be very thin Sections can be thicker than with TEM

Exam Tip: As with any exam question, if you are asked to compare or contrast, make
sure you state both sides of the comparison.

Graticule and stage micrometer

An eye piece graticule is used in microscopy to measure the size of objects viewed
through a microscope. The graticule cannot be use alone and needs to be coupled with a
stage micrometer to calibrate the graticule every time you change magnification on your
microscope.

You will carry out this practical in your required practical 2

An example of how it works (but you will understand this when you carry out this practical):

Calculating the size of cellular structures

Image size
Magnification = Image Actual

I
Actual = Image
Magnification
 Image = Actual x Magnification MA

Magnification
Actual size

When asked to calculate the actual size or magnification of a cellular structure in an exam
there are a few simple rules to follow.

Rule 1
If required rearrange the formula, Image = Actual x Magnification as shown

above. Note: It’s easy to remember the formula as I am i.e. I = a x m

Rule 2
If required it is often easier (although not essential) to make any measurements in mm.

This allows for easier conversion to micrometres (µm) = 10-6 metre which is the unit most
often used in measuring the size of cellular structures.

Rule 3
Ensure you convert any measurements you make (or any the examiner may provide) to
the same units required for the answer e.g. convert millimetres (mm) into micrometres
(µm).

It is often better to do this before carrying out the calculation.

Units of Measurement
1 nm (nanometre) = 10-9 metre
-2
1 cm (centimetre) = 10 metre
Make sure you know these
-3
1 mm (millimetre) = 10 metre measurements and can convert
one into the other.
1 µm (micrometre) = 10-6 metre

Converting units

X1000 x1000

mm µm nm

/1000 /1000

Exercise
Q measurement Convert into Answer

1 10 cm mm 100 mm
 2 650 mm µm

3 17 mm nm

4 25.5 nm mm

5 398 µm mm

6 10 cm µm

7 5.5 nm µm

8 1.5 m µm

For example

You are asked to calculate the actual size of an organelle (e.g. a chloroplast) in
micrometres (µm) = 10-6 metre. You are given the magnification as x 30 000

Rule 1 – Rearrange the formula

Actual size = Image size

Magnification

Rule 2 – Measure the image size e.g. = 90 mm

Rule 3 – Convert to units required in the answer i.e.

micrometres 90 mm = 90 x 103 (i.e. x 1000) micrometres (µm) =

90 000

Therefore Actual size = Image size = 90 000 µm = 3.0 µm Magnification

30 000
The Cell-surface Membrane

The entry into and exit of substances out of cells is controlled by the cell-surface
membrane or plasma membrane, which surrounds the cytoplasm of a cell.

All cells have a cell-surface membrane and, in addition, eukaryotic cells have internal
membranes. The basic structure of these plasma membranes is the same and enables
control of the passage of substances across exchange surfaces by passive or active
transport.

The cell surface membrane consists mainly of a phospholipid bilayer (double layer) and
protein. The structure of the cell-surface membrane is described as the fluid-mosaic
model as:

∙ the phospholipid molecules in the bilayer are constantly moving about giving a fluid
structure.
∙ the protein molecules are unevenly distributed throughout the membrane forming a
mosaic.

The partial permeability of the cell membrane is related to the type and distribution of
protein and phospholipid molecules present in the membrane as described below.

∙ The hydrophilic heads of the phospholipid bilayer are orientated either inwards toward
the cytoplasm or outwards toward the watery extra-cellular fluid.

∙ The hydrophobic tails (fatty acid chains) of the phospholipid molecules in the bilayer are
attracted towards each other.

∙ Very small molecules such as carbon dioxide and oxygen diffuse through the gaps
between phospholipids. Water passes through water protein channels (aquaporins) by
osmosis.

∙ The phospholipid bilayer enables lipid soluble molecules to pass quickly through the
cell membrane but restricts passage of water soluble ions and polar molecules.

Molecules that have little or no charge Charged substances cannot cross the
or/and are very small can pass between phospholipid bilayer by simple diffusion
phospholipids. and require transport proteins.
∙ Cholesterol provides strength to the membrane and restricts the movement of
phospholipids so that the membrane is less fluid and less ions are loss from the cell.#

∙ Unsaturated fatty acids increase the permeability of the membrane whereas saturated
fatty acids decrease its permeability

∙ Protein molecules spanning the membrane act as carriers or channels which aid the
passage of water soluble ions and polar molecules across the membrane

∙ Carrier Proteins possess a specific tertiary structure complementary to polar molecules

e.g. glucose or amino acids, aiding their transport by facilitated diffusion and active
transport.

∙ Channel Proteins also have a specific tertiary structure, which together with its charge
and size determines which molecules can be transported across the membrane by
facilitated diffusion.

∙ Other embedded protein molecules are important in ‘cell signalling’ and act as specific
receptors for hormones e.g. insulin, which attach to the membrane allowing the cell to
respond. These receptor proteins have a specific tertiary structure allowing a specific
 hormone with a complementary shape to attach to its binding site.

∙ Branched carbohydrate (sugar) chains stick out from the outer surface of some
membranes like antennae. They are attached to lipids, forming glycolipids, or to
proteins forming glycoproteins. As well as being able to act as receptors,
glycoproteins can be involved in cell-cell recognition as they can act as antigens,
therefore foreign antigens (e.g. on invading bacteria) can be recognised and attacked
by the immune system.

Microvilli
∙ Microvilli are finger like extensions of the cell surface membrane of some animal
cells.

∙ They increase the surface area and are particularly numerous on cells specialised
for absorption such as epithelial cells in the small intestine.

∙ The increase in surface area also improves efficiency of digestion in the gut
because certain digestive enzymes are attached to their surface.

∙ The microvilli can be seen with a light microscope as a fringe across the top of the
cell called a brush border.
Transport across membranes
Movement of substances into and out of cells through the plasma membrane can
occur by diffusion, facilitated diffusion, osmosis, active transport and co
transport (see topic 3 notes for co transport).

Diffusion
Diffusion is the net movement of molecules from a high concentration to a low
concentration until the molecules are equally distributed.

Diffusion is a passive process i.e. it does not require energy from respiration.
Gaseous exchange occurs via diffusion.

The rate of diffusion across a membrane occurs at a rate determined by a number of

factors.
 Rate of diffusion is proportional to Surface area x Concentration difference
(gradient) Thickness of exchange surface

The rate of diffusion is increased by a higher concentration gradient, a large surface

area e.g. microvilli, and by a short diffusion distance e.g. a single cell layer.

Facilitated diffusion
∙ This
process allows the transport of polar molecules such as glucose and amino acids
across membranes.

∙ It involves the use of channel proteins and carrier proteins

∙ Facilitated diffusion is a passive process i.e. it does not require energy. ∙

It is not inhibited by respiratory inhibitors such as cyanide.

Carrier proteins.
Channel proteins.

Active Transport
Active transport is the movement of molecules or ions through a partially permeable
membrane by carrier proteins against a concentration gradient.

∙ Active transport requires energy from the hydrolysis of ATP produced during
respiration.

∙ The molecule attaches to the carrier protein which has a specific tertiary structure
complementary to the polar molecule e.g. glucose, amino acid, being transported.

∙ Thebinding causes the carrier protein to change shape and transport the molecule
across the membrane (see diagram below).
 Factors which decrease the rate of respiration will therefore decrease active
transport: 1) Lowering of temperature.
2) Lack of oxygen.
3) Metabolic and respiratory inhibitors e.g. cyanide

∙ Cells involved in active transport possess a large number of mitochondria to provide

the ATP required via respiration.

Note – Cells may be adapted for rapid transport across their internal or external
membranes by an increase in the number of protein channels and carrier molecules
in their membranes.
Osmosis
Osmosis is the net movement of water molecules from a dilute solution to a concentrated
solution across a partially permeable membrane.

Osmosis is essentially the diffusion of water molecules across a partially (selectively)

permeable membrane.
 ∙ In terms of solute concentration - osmosis is the movement of water from a low
concentration of solute molecules to a high concentration of solute molecules.

∙ In terms of water concentration - osmosis is the movement of water from a high

concentration of water molecules to a low concentration of water molecules.

Water potential
Water potential can be defined as the potential (tendency) of water molecules to leave a
solution by osmosis.
The greater the concentration of water molecules in a cell or solution the higher is its water
potential.

Osmosis is the net movement of water molecules from a higher (less negative) water
potential to a lower (more negative) water potential through a partially permeable
membrane.
 Solutions A and B separated by a partially permeable membrane
Solution A Solution B

20 % Glucose 70 % Glucose

Lower solute concentration Higher solute concentration

Higher water potential Lower water potential

∙ Solution A has a higher water potential is as it has a lower concentration of solute molecules
and therefore a higher concentration of ‘free’ water molecules.

∙ Solution B has a lower water potential as it has a higher concentration of solute molecules and
a lower concentration of ‘free’ water molecules.

∙ Water movement would occur by osmosis from solution A (higher water potential) to solution B
(lower water potential).

∙ Net water movement would continue until the solutions were isotonic i.e. of equal water
potentials.

∙ At this point water movement would still occur, but would be equal in both directions, i.e. there
would be no net movement of water.
 The diagram below shows three plant cells with different water

potentials. The arrows indicate the movement of water by osmosis between these cells.

-350Ψ

-300Ψ

-375Ψ
Osmosis and cell turgor
∙ Water movement occurs along a water potential gradient.

∙ The presence of ions in a cell lowers its water potential and water can enter via osmosis.

∙ As water enters the cell vacuole enlarges and the vacuole, cytoplasm and cell membrane
exert an outward turgor pressure on the cell wall.

∙ Cell turgor is essential in providing support in many plants.

The picture below shows how a plant cell plant cell responds when placed in distilled water (and
a concentrated solution

Osmosis in red blood cells

∙ If a red blood cell is placed in an isotonic solution (i.e. equal water potential), osmosis does
not occur as there is no net movement of water molecules.
∙ If the cell is placed in a solution with a low solute concentration (i.e. a higher water potential),
osmosis occurs and water moves in down the water potential gradient from high to low. The
cell will swell as its volume increases and the cell may burst. The cell may burst when the
pressure increases to such an extent that the cell surface membrane ruptures.

∙ If the cell is placed in a solution with a high solute concentration (i.e. a lower water potential),
osmosis occurs and water moves out of the cell down the water potential gradient. The cell
will shrink as its volume decreases.
Cell division
DNA, Genes and Chromosomes
DNA

In prokaryote cells, DNA molecules are short, circular and are not associated with protein.
Prokaryotes do not form chromosomes

In the nucleus of eukaryote cells, DNA molecules are very long, linear and associated with
proteins called histones. Together a DNA molecule and its associated proteins form a
chromosome.

The mitochondria and chloroplasts of eukaryotic cells also contain DNA which, like the DNA
of prokaryotes, is short, circular, is not associated with protein and does not contain introns.

Chromosomes

During cell division in eukaryotes, DNA and histones are organised into structures called
chromosomes.

Genes are located along chromosomes

1. A gene occupies a fixed position, called a

locus on a particular DNA molecule or
chromosome.

2. A gene can exist in different forms called

alleles that code for different types of the
same characteristic.

3. Alleles of a particular gene are located in

the same relative position (locus) on
homologous chromosomes.

4. A homologous pair of chromosomes are

the same length and carry genes
controlling the same characteristics but
not necessarily the same alleles.

5. A homologous pair of chromosomes

consists of a maternal and a paternal
chromosome.
Mitosis
Mitosis is a type of nuclear division which produces
cells that are genetically identical. During mitosis the
parent cell divides to produce two daughter cells, each containing an identical copy of the DNA
in the parent cell. (Meiosis is the other type of nuclear division in which four genetically
different cells are produced from the parent cell – see later notes).

∙ Mitosis increases cell number for growth and repair of tissues.

∙ During mitosis, the nuclear material becomes organised into structures called chromosomes.

∙ In a normal body cell, the chromosomes can be grouped into homologous pairs of
chromosomes.

∙ Diploid number (2n) represents the total number of chromosomes in a normal body cell. In
man this is 46 i.e. 23 homologous pairs.

∙ Haploid number (n) represents a single set of chromosomes i.e. one member from each
homologous pair. In man this is 23, the number of chromosomes in a gamete (sperm or
ovum).

∙ Mitosis produces cells with the same number of chromosomes as the parent cell so that a
diploid parent cell will divide to produce two identical diploid cells.

The Cell cycle

Mitosis is part of the cell cycle in which cells undergo a regular cycle of nuclear and cell
division separated by periods of cell growth. The cell cycle has three stages.

1. Interphase - this represents the non-dividing cell when cell growth

occurs. 2. Nuclear division - the nucleus divides into two (mitosis)
3. Cytokinesis –the cytoplasm divides into two (mitosis).

As shown above, interphase often represents the longest time period of the cell cycle. However,
in rapidly dividing cells (e.g. those lining the intestines) the time a cell spends in interphase is
short enabling rapid multiplication and replacement of cells. Note: Not all cells in an organism
are capable of cell division.
Interphase

The cell is carrying out its normal cellular functions but during late interphase it prepares for
nuclear division in the following ways.
1. Increase in protein synthesis.
2. DNA content is doubled via DNA
replication.

3. Cell organelles are replicated e.g.

mitochondria and ATP content is
increased, as cell division is an active
process.

Mitosis
Although mitosis is a continual process, biologists divide it into four stages, prophase,
metaphase, anaphase and telophase.

Prophase

1. Chromosomes form. Due to DNA replication during interphase each chromosome

consists of two identical sister chromatids joined together by a centromere.

2. Each chromosome then shortens and thickens. .

3. The centrioles move to opposite poles (sides) of the cell (centrioles are not present in
plant cells.

4. The nuclear membrane breaks down.

Metaphase

1. The centrioles form a spindle across the cell. The spindle consists of protein
microtubules.

2. Each chromosome moves to the equator (centre) of the spindle and attaches to it via
its centromere. Sister chromatids are orientated towards opposite poles of the cell.
Anaphase
1. The centromere splits and the sister chromatids separate.

2. Sister chromatids are pulled to opposite poles of the cell by the spindle microtubules.

Telophase

1.The chromatids are at opposite poles of the cell and begin to uncoil. The nuclear membrane
reforms.

2. The two cells are genetically identical to each other and to the original parent cell.

Cytokinesis

This follows nuclear division and involves the splitting of the cytoplasm into two. Two new cells
form as a cell-surface membrane and in plant cells, a cellulose cell wall forms.

Summary of Mitosis

Put a tick in the box to indicate at which stage of the cell cycle each of the following events
occurs:
Event I P M A T
1 Spindle fibres form

2 DNA replication occurs

3 Sister chromatids are pulled to opposite poles

4 Nuclear membrane breaks down

5 Nuclear membrane reforms

6 Chromosomes shorten and thicken

7 Chromosomes move to the equator of the cell

8 Cell organelles are replicated

9 DNA polymerase is active

10 In animal cells, centrioles move to the poles

Cancer and the cell cycle

Cancer is a group of diseases caused by uncontrolled growth and rapid division of cells. This
often results from damage to the genes that regulate mitosis and the cell cycle.

Uncontrolled cell division and growth results in a group of abnormal cells, called a tumour.
Cells may break away and move to other areas of the body leading to spread of a cancer.

Cancer treatments often use drugs to stop cancerous cells dividing. Drugs may be used to
inhibit the enzymes, DNA helicase or DNA polymerase (both important in DNA replication) or to
inhibit the formation of the spindle.
Preparation of stained squashes of cells from plant root tips; setup and use of an optical
microscope to identify the stages of mitosis in these stained squashes and calculation of
a mitotic index.

For the majority of the cell-cycle, the genetic material of a cell is present as chromatin and
individual chromosomes are not visible.

The best chance of

seeing cells in stages of mitosis is to examine actively growing plant tissue. This means either
root tips or shoot tips. Usually growing onion or garlic roots are used. Notes on the Method
Like almost all biological specimens, these cells will not be visible unless stained.
The hydrochloric acid weakens the cellulose walls of plant cells.

This ensures that the stain can penetrate the cells and allows the tissue to be squashed out one
cell thick.

The preparation needs to be only one cell thick so that:

∙ When under the microscope, light can pass through the sample.

∙ So that the chromosomes of one cell can be clearly seen, without other cells being on top
or beneath them.

A mitotic index is the ratio of the number of cells in a tissue undergoing mitosis to the total
number of cells.

Mitosis questions

Stained onion root tip cells were observed under an optical microscope at a x400 magnification.
The student counted the cells at each of the different stages of mitosis and recorded the
information in the table shown below.

(a) Complete the column to show the % of cells at each stage.

Stage of mitosis Number of cells at % of cells at this stage
this stage

Interphase 80

Prophase 10

Metaphase 16

Anaphase 18

Telophase 8

(b) One complete cell cycle takes 24 hours. The number of cells at each stage is
proportional to the time spent at that stage. Calculate the length of time spent at
interphase. Show your working.
The table below shows the average duration of each stage in a grasshopper embryo.
Stage of mitosis Mean duration/minutes

Interphase 20

Prophase 105

Metaphase 13

Anaphase 8

Telophase 54

(c) Give one piece of evidence from this table which indicates these cells are dividing
rapidly.
..................................................................................................................................
.
..................................................................................................................................

(d) Calculate the mitotic index for the cells below:

Binary fission

Prokaryotes (Bacteria) mainly reproduce by asexual reproduction by a process called binary

fission. This means that one cell divides into two daughter cells.

This involves:

∙ Cell elongation

∙ Replication of the circular DNA and of plasmids

∙ A septum (wall) starts to be synthesised to divide the cell.

∙ The septum (wall) grows right across the cell which causes division of the cytoplasm to
produce two daughter cells, each with a single copy of the circular DNA and a variable
number of copies of plasmids.

∙ Finally, the two new daughter cells separate and begin a new cell cycle.
From a single cell, binary fission forms two cells, then four cells, then eight, sixteen, thirty-two
and so on. The time taken for a bacterial population to double in number is called the
generation time. In ideal conditions some bacteria can divide in 20 minutes, though for many
species it is 15-20 hours.

This continued doubling of numbers is called an exponential increase in population. The

population descended from each cell after a number of generations of growth in favourable
conditions can be estimated by the equation:
Population in the nth generation = 2n

So, after four generations the population will be

24= 2 x 2 x 2 x 2
= 16 cells.
Calculations

1. If bacterium X has a generation time of 20 minutes, how many bacteria will be produced
after 3 hours?

2. 10 bacteria are left to grow in a beaker. The generation time for this bacterium is 15
minutes. How many bacteria will be produced after 2 hours?

Pathogens
Pathogens include disease-causing microorganisms. Infectious pathogens include bacteria,
viruses and fungi.

Disease occurs when an infection leads to recognisable symptoms in the host. When a
pathogen is transferred from one individual to another, it is known as transmission.

Defence mechanisms

Non-specific defence mechanisms

These mechanisms respond to all types of pathogens in the same way. These are general
defence mechanisms and include barriers to entry e.g. the skin, mucus and cilia, and non
specific responses such as phagocytosis.

Specific defence mechanisms

These mechanisms involve a specific response against the pathogen. These responses often
provide long-lasting immunity and involve B cells and T cells. These are both types of a white
blood cell known as a lymphocyte.

There are two specific defence mechanisms.

∙ Humoral response involving B cells (B lymphocytes)

∙ Cellular response involving T cells (T lymphocytes)

Note: For the exam you only need to use B cells and T cells. The term lymphocyte is
not required but you will see it in lots of textbooks.
Non-specific defence mechanisms
Mucus
Secreted by the epithelial cells lining the nasal passages and the respiratory tract.
Mucus is ‘sticky’ and traps bacteria and dust particles entering the air passages.

Cilia
These are minute hairs on the epithelial cells in the upper parts of the respiratory
tract. Cilia beat to move the mucus and trapped particles to the throat. Swallowing
carries the pathogens to the stomach where they are killed by stomach acid or
enzymes

Phagocytosis (‘cell eating’)

This is the engulfment and destruction of microorganisms by phagocytic white

blood cells.

The phagocyte extends around the pathogen and engulfs it forming a phagosome or
phagocytic vesicle.

A lysosome (cell organelle) containing lysozymes (hydrolytic enzymes) fuses with

this phagosome and the enzymes hydrolyse the pathogen.
Soluble digested products are absorbed whereas indigestible material is removed.

Phagocytes destroy microorganisms that enter the blood and other tissues helping to
reduce their spread to other parts of the body.

Phagocytes can also act as ‘antigen-presenting cells’. This involves the phagocyte
removing the antigens from the pathogen they destroy and presenting the antigens
on their cell-surface membrane to T cells (see later notes on the cellular response).

Phagocytosis

A non-specific defence
mechanism
Specific defence Mechanisms

These mechanisms involve the immune system and a specific response against a pathogen,
abnormal cell or toxin. These responses often provide long-lasting immunity and involve B
lymphocytes and T lymphocytes. These are both types of white blood cell.

There are two specific defence mechanisms.

∙ Humoral response involving B lymphocytes

∙ Cellular response involving T lymphocytes

Specific defence mechanisms involve the activation of B lymphocytes (humoral response) or T

lymphocytes (cellular response) against antigens which have entered the body.

Antigens

Antigens are proteins or glycoproteins that appear ‘foreign’ to the individual organism
exposed to them. Antigens stimulate the production of antibodies by B lymphocytes.

Antigens may be present:

∙ on the surface of a pathogen

∙ on the cell-surface membrane of other organisms of the same species e.g. organ transplant ∙
abnormal body cells e.g. cancer cells
∙ as a toxin e.g. a free molecule often produced by a pathogen
The Humoral Response

This involves B cells and the production of antibodies in response to a specific antigen e.g. a
protein on the surface of a pathogen

When exposed to the appropriate antigen, B cells secrete the specific antibody into the blood
plasma to destroy or neutralise the antigen and the pathogen.

Structure and Function of Antibodies

Antibodies are proteins found in blood plasma, tissue fluid and breast milk.

The basic structure of an antibody molecule consists of four polypeptide chains – 2 heavy and
2 light chains – joined together by disulfide bonds.
Each polypeptide chain consists of a constant region and a variable region.

∙ In the constant region the sequence of amino acids is the same in all molecules of the
same type of antibody.

∙ In the variable region the amino acid sequence is different in different molecules of the
same type of antibody.

Antigen –binding
site

disulfide bond

The variable regions of the heavy and light chains form two antigen-binding sites.

These sites have a specific tertiary structure complementary to the structure of the antigen
molecule to which they attach (similar to a lock and key) to form an antibody-antigen
complex..

Note: Never use the term active site to describe an antigen-binding site. Only enzymes
have an active site.
Antibodies do not directly destroy the antigen/pathogen. However, the formation of the
antibody-antigen complex stimulates different processes that do cause the destruction of the
pathogen. Two of these processes are:

∙ Agglutination of antigens
∙ Stimulation of phagocytosis

Agglutination

Agglutination refers to the ‘clumping’ together of cells possessing the antigen against which
specific antibodies (agglutinins) react. An antibody molecule can use its two antigen-binding
sites to attach to the same antigen present on two different cells.

This joins the cells together. As more antibody molecules attach more cells are linked together
to form an agglutinated mass (clump) of cells which are then more easily destroyed e.g. by
phagocytosis.

Agglutination of donor red blood cells occurs when an individual receives the wrong blood
group.

Stimulation of phagocytosis
One type of antibody attaches to the antigen on the surface of a pathogen and identifies it for
destruction by phagocytic white blood cells.

Phagocytes have receptors in their cell-surface membranes that recognise the antibody, and
enable them to bind to, engulf and then destroy the pathogen.

Production of antibodies in the humoral response

1. The body has a large number of different types of B lymphocytes (B cells), each type
capable of producing a different specific antibody.
 2. These B lymphocytes secrete small amounts of their specific antibody onto their cell
surface membrane.

3. If a specific antigen e.g. on a pathogen, attaches to the complementary antibody on B

lymphocytes, these B cells are stimulated to divide by mitosis. This results in a large
population of identical plasma cells i.e. a clone of plasma cells (known as clonal
selection and expansion). Helper T cells (TH cells) are also involved in this process.
TH cells stimulate B cells to divide to form plasma cells and to secrete their specific
antibodies.

4. The plasma cells will all produce the same specific antibody and secrete it into the blood
plasma. Some of the B cells are stimulated to divide and develop into memory B cells.

5. The antibodies bind specifically (lock and key) to the antigens forming an antibody
antigen complex. This stimulates processes leading to the antigen/pathogen being
destroyed.
Note: This first, primary response is relatively slow requiring up to 72 hours to produce
significant amounts of antibody. During this time the microorganisms reproduce and disease
symptoms may arise.

6. If the same antigen (pathogen) is subsequently encountered these memory B cells divide
and develop into plasma cells. These plasma cells secrete antibodies quicker and at a
higher concentration (secondary response) than in the primary response. This provides
immunity as the microorganisms are destroyed before disease symptoms develop.

Antigenic variation

Some microorganisms e.g. the influenza virus have high a mutation rate and this leads to
antigenic variation. Therefore, even though an individual may become ‘immune’ to one strain
of the virus, this will not provide immunity to the new forms as they are not recognised by the
memory B cells and the antibodies previously produced are not complementary.
Summary of role of B cells in humoral immunity
 Cellular Response

This involves T cells. They do not produce antibodies but they do possess proteins (T cell
receptors) on their cell surface membrane which recognise specific antigens. The receptors on
each T cell respond to a single specific antigen.

1. There are a vast number of different types of T cell, each one responding to a different
antigen.

2. The antigen is presented to a helper T cell (TH cells) by an antigen presenting cell i.e. a
phagocyte. After the phagocyte has engulfed the pathogen, it removes its antigens and
embeds them in its cell-surface membrane. Helper T cells with the complementary
protein receptor will bind to the antigen.

3. This stimulates the TH cells to divide by mitosis and form a clone of genetically identical
T cells all with the same receptor. The cloned TH cells:

∙ activate cytotoxic T cells (TC cells) – which attach to the specific antigen on the
pathogen/‘foreign cell’ and secretes chemicals (e.g. a protein called perforin) to destroy
it.

∙ develop into more helper T cells (TH cells) which stimulate B lymphocytes to divide into
plasma cells and secrete antibodies.

∙ develop into memory T cells – which remain in the blood after the infection has cleared
and produce a quicker response (secondary response) if a future infection occurs with
the same antigen/pathogen.

Summary of role of T cells in the cell mediated response

Summary of the humoral and cellular responses
 Types of Immunity

There are two main types of immunity – passive immunity and active immunity.
1. Passive Immunity

This is where an individual receives pre-formed antibodies from an outside source. The
individual is not exposed to the antigens and does not form antibodies or memory cells.
Although antibodies will be immediately present, this only provides short-term immunity as the
antibodies are not produced by the individual and are not replaced once they are broken down.

∙ Natural Passive Immunity - the antibodies are obtained across the placenta and through
breast milk. This provides short-term protection, as the body is not stimulated into
producing its own antibodies and memory cells.

∙ Artificial Passive Immunity - the pre-formed specific antibodies are injected usually
following exposure to particularly infectious pathogens or toxins e.g. the rabies virus,
snake venom (toxin). Again, this provides short-term protection.

2. Active Immunity

This is where an individual exposed to the antigen produces antibodies and memory cells.
Although it takes time to produce these antibodies, the immunity is usually long-term as the
immune system has produced its own antibodies and memory cells.

∙ Natural Active Immunity - this results from an individual becoming infected e.g. chicken
pox, and is exposed to the specific antigen. Long-term immunity develops as memory
cells are formed.

∙ Artificial Active Immunity - this results from an individual being vaccinated

(immunisation) and the immune system produces its own antibodies and memory cells
providing long-term immunity. (see below)
Vaccination

A vaccine contains antigens from a specific pathogen. The antigens in the vaccine may be
present in the form of the dead pathogen, a weakened strain of the pathogen or just antigens
(removed from the pathogen).

Injection of this vaccine stimulates an immune response with the production of plasma cells,
which release specific antibodies, memory B cells and memory T cells. These memory cells
provide long-term immunity.

Note: Sometimes a ‘booster’ injection of the same antigen is given at a later date, to ensure a
more effective response (secondary response) due to exposure to more antigen producing
long term, often life-long immunity due to the memory cells formed.
Herd immunity

The higher the percentage of the population vaccinated against a particular infection the less is
the risk of an epidemic. (Epidemics involve the wide spread transmission of the disease through
the population). This is because the probability of encountering an unprotected individual will be
low.

Some parents do not vaccinate their children for a variety of reasons and thus there is a
susceptible part of the population where the infection can reside.

A very important factor in determining whether a child is vaccinated against a particular infection
is the possibility of side effects and the effectiveness of the vaccine in providing protection.

More recently, one study indicated a link between the MMR (measles, mumps, rubella) vaccine
and autism. Despite the findings of this research proving to be unreliable a number of parents
did not vaccinate their children with the MMR vaccine. This led to an increase in the number of
cases in the UK.
HIV and AIDS

Acquired immune deficiency (AIDS) syndrome is caused by infection with the human
immunodeficiency virus (HIV).

∙ HIV is a retrovirus containing RNA and the enzyme reverse transcriptase which produces
DNA in the host cell using RNA as a template.

∙ The RNA and enzyme are surrounded by a capsid (protein coat).

∙ The capsid is surrounded by a lipid envelope which contains glycoprotein ‘spikes’. Specific
glycoproteins on the surface of the virus enable it to attach to its host cell, helper T cells (TH
cells).

Human immunodeficiency virus (HIV)

Transmission of HIV

HIV is transmitted only by the introduction of blood, semen or vaginal secretions from an
infected individual into the bloodstream of another individual. This can occur by:

∙ Sexual transmission

∙ Blood products / transfusions of infected blood

∙ Sharing of needles e.g. drug users.

∙ Mother to baby (via placenta, during child birth or via breast milk)
HIV replication

∙ Viruses attaches to CD4 helper T cells by glycoproteins (gp120) on their surface

binding specifically to receptor sites on the membrane of the helper T cells
(lymphocytes).

∙ Viral RNA and the enzyme reverse transcriptase are released and enter the T
lymphocyte.

∙ In the helper T cell viral DNA is formed by the enzyme reverse transcriptase using the
viral RNA as a template.

∙ Viral DNA enters the nucleus and attaches to host DNA

∙ Viral DNA replicates with host DNA

∙ The viral DNA may remain inactive (latent) for up to 10 years

∙ When activated the viral DNA controls the synthesis of viral proteins and viral RNA
within the T cell

∙ HIV particles are assembled and the T helper cell is destroyed as viruses are
released. These viruses infect other T helper cells. As the viruses reproduce the
 number of T helper cells (lymphocytes) in the blood decreases.

∙ T helper cells (lymphocytes) are essential in stimulating antibody production by B

lymphocytes in the immune response to combat infections.

∙ As the immune response breaks down an individual suffers from multiple opportunistic
infections and / or tumours that eventually lead to death. Two common opportunistic
diseases are Kaposi’s sarcoma and pneumonia.
Symptoms

Four phases can be distinguished

∙ During the first phase after infection the body produces HIV antibodies and there is a short
flu-like illness. A skin rash and swollen glands sometimes occurs.

∙ The second phase is the antibody-positive phase (HIV positive phase) – the period between
infection and the onset of clinical signs – may last from a few weeks to 13 or more years.

∙ The third phase is the AIDS-related complex (ARC). The individual may contract a variety of
opportunistic bacterial, viral and fungal infections – at this stage not life threatening. Loss of
weight and a reduction in the number of T helper cells occurs.

∙ The fourth phase involves opportunistic infections of the body organs, the development of
secondary cancers and HIV wasting syndrome with dramatic weight loss. Most AIDS
patients eventually die from pneumonia as the immune system collapses.

Note: Do not learn the time span of these phases. You

only need to know the main symptoms and these
time spans can vary considerably if the patients are on
anti-viral drugs which help to prevent the virus
replicating.
Preventing HIV infection

∙ Reduced promiscuity and ‘safe’ sex via the use of condoms to prevent the
transmission of semen and vaginal fluids.

∙ Blood screening to prevent HIV antibody positive blood or blood products e.g. factor 8
being passed on.

∙ Tissue screening for transplants – to prevent donations from HIV antibody individuals.
 ∙ Provision of clean needles for drug users to prevent sharing of used needles and
transmission of blood products. Careful regulation of tattooing where reused needles
can transmit the virus.

∙ Bottle feed instead of breast feed and elective caesareans.

Treatment
HIV is a viral disease and therefore antibiotics are ineffective. Antibiotics are only effective
against bacteria either inhibiting their division or destroying them. Antibiotics affect cellular
structures of bacteria such as the cell wall and ribosomes. As viruses do not possess cellular
structures (they use those of their host cell), antibiotics are completely ineffective against
viruses.

Several drugs have been developed to prevent the replication of the virus including reverse
transcriptase inhibitors.

Researchers are working on the production of a vaccine but no product is yet available for
testing on humans. The ability of the virus to mutate and alter the proteins on its surface
(‘antigenic variation’) is a major problem in developing the vaccine.

Monoclonal Antibodies

Monoclonal Antibodies

These are identical antibodies, having the same tertiary structure. Therefore, they have the
same antigen-binding site. These are produced in large amounts from a single clone of
antibody-producing B cells.

Note: For the exam you do not need to know the method by which monoclonal antibodies are
produced.

Uses of Monoclonal antibodies

1. To target medication to specific cell types by attaching a therapeutic drug to an antibody.
E.g. cancer cells display different antigens to healthy cells, so they can be targeted by
monoclonal antibodies with a toxic drug attached. This means cancer cells are killed and
healthy cells are unaffected.

2. For medical diagnosis. For example:

∙ to detect the presence of specific antigens (i.e. pathogens) in body fluids to diagnose if a
person is infected with a particular disease

∙ to detect the presence of specific antibodies produced by a person against an antigen

(i.e. pathogen)

The ELISA test (see following notes) is an important method by which medical diagnosis can
be carried out.

Note: For the exam you will also need to be able to:

∙ discuss ethical issues associated with the use of vaccines and monoclonal antibodies

∙ evaluate methodology, evidence and data relating to the use of vaccines and monoclonal
antibodies.
The ELISA test

The ELISA test is an enzyme linked immunosorbent assay i.e. a method of measuring the
amount of antigen (Direct ELISA test) or the amount of antibody (Indirect ELISA test) in a
sample. It involves using monoclonal antibodies, some with attached enzymes that catalyse
easily detectable reactions.

The Direct ELISA test is used to detect antigens, therefore it can determine if a pathogen is
present in a sample.

The Indirect ELISA test is used to detect antibodies, therefore it can determine if an individual
has antibodies against a pathogen, indicating a previous or current infection.

The following is one method for a Direct ELISA test

Stage 1
∙ The test is performed in a plastic tray divided into wells, each of which has been coated
with a specific monoclonal antibody (produced specifically against the antigen of the
pathogen to be detected).

∙ This monoclonal antibody is irreversibly bound to the plastic surface of the well.

Stage 2
∙ The fluid sample to be tested (e.g. blood, urine) is added to the well.

∙ If molecules of the specific antigen are present, they will bind to the monoclonal antibodies
to form antibody-antigen complexes.
Stage 3
∙ A second monoclonal antibody (also specific for the same antigen), which has an
enzyme attached, is added to the well.

∙ The second antibody can only bind to the specific antigen to form an antibody-antigen
complex - it cannot bind directly to the first antibody or the plastic.

∙ The well is then washed to remove any unbound second antibody so that any second
antibody (with the attached enzyme) will only remain in the well if the specific antigen is
present and the second antibody attaches to it.

Stage 4
∙ A suitable substrate (usually colourless), for the enzyme (attached to the second antibody)
is then added. If the enzyme is still present in the well it will convert the colourless
substrate into a coloured product.

∙ The coloured product will only be formed when the enzyme remains in the well showing
that the second antibody attached to the specific antigen. This will indicate a positive
result for the detection of the antigen (pathogen) in the sample being tested.

Note: The well will remain colourless if the specific antigen is not present as all the second
antibody (with attached enzyme) will be washed away during step 3. Therefore, no enzyme will
be present to convert the colourless substrate into a coloured product.
Direct ELISA test

Note: Washing of the wells can be done at each stage as shown in the diagrams however it is
the final washing of the well which is important in preventing ‘false positives’.

Indirect ELISA test

The amount of colour produced in each well is determined by the amount of enzyme present.
This in turn is proportional to the amount of antigen extracted from the test sample.

A calibration curve can be produced using known amounts of antigen to determine the precise
amount of antigen (or antibody – indirect ELISA) in a sample.

Ethical issues associated with the use of vaccines and monoclonal

antibodies Vaccines

Use of animals in the production of vaccines

Side effects leading to long term issues (can this be balanced by the greater risk of harm by
catching the disease in question).
Will it impact the natural immunity in communities leading to greater harm (malaria

vaccination). Monoclonal antibodies

The production of monoclonal antibodies involves inducing cancer in mice, as both antibody
producing cells and tumour cells are required to produce monoclonal antibodies. Although
there have been some great results in the treatment of some cancers, there have been some
serious side effects and deaths with the use of monoclonal antibodies in treatments.