Biological Basis of Oral Sciences .pptx

Biological Basis of
Oral Sciences
AMOS, session 2022/2023
13th April 2023
Bt Ken Wong

Learning Outcomes
1. Describe the structure and functions of cell membrane.
2. Describe and compare between these three types of
microscopes, compound light microscope, transmission and
scanning electron microscope.
3. Describe the differences between prokaryote and eukaryote
cells.
4. Describe the structure and function of these organelles.
5. Compare between passive transport and active transport.
6. What are stem cells?
7. Describe totipotency, pluripotency, unipotency and
multipotency.
8. What is ECM? Describe the composition of ECM.
9. Describe scurvy, osteogenesis imperfecta, Ehlers-Danlos
syndrome, Marfan syndrome and α1-antitrypsin deficiency.
10. What is cell adhesion? Describe all cell adhesion molecules.
11. Explain some examples of defects in adhesion molecules.
12. Explain what happens when there an increase of adhesion
molecule expression and inflammation.
13. Using a diagram, explain the signalling mechanism of G protein
signalling.
14. What are Ras G proteins? How is this signalling mechanism
activated? What happens when there is mutation to this
signalling?
15. Explain the stages of cell division and cell cycle. What is
Hayflick’s limit?
16. How is the assessment of cell proliferation carried out?
17. What are the cell cycle regulators?
18. Explain tumour suppressors and checkpoints.
19. What happens when DNA damage occurs?
20. Explain the basis of some chemotherapeutic agents specific to
cell cycle.
21. Define necrosis and apoptosis.
22. What is the function of apoptosis.
23. Explain the mechanism of apoptosis with a diagram.
24. Explain apoptosis in cancer, autoimmune conditions and
neurodegenerative illnesses.
25. Explain four laboratory methods to assess apoptosis.

Structure and Functions
of Cell Membrane
Major components
Structure

M a j o r C o m p o n e n t s o f C e l l u l a r M e m b r a n e
Lipid Protein

Structure
of Cell
Membrane
• Basic structure = phospholipid bilayer
• 2 antiparallel sheets of phospholipids form the membrane
• Surrounds the contents of the cell
• Inner leaflet = closest to cytosol
• Outer leaflet = closest to exterior environment
• Cholesterol fits between phospholipid molecules
• Proteins also a/w membrane
• Enable the biological functions according to the need of the
particular cell

Structure of Cell Membrane
Bilayer arrangement
Asymmetry
Fluid mosaic model
Lipid rafts

Bilayer Arrangement
• Hydrophobic fatty acid tails facing away from the polar,
aqueous fluids of both the cytosol and the environment
(such as blood or other cellular fluids including lymph)
• Hydrophilic head oriented toward the polar environment.
• Phospholipids of each layer are opposite to each other
• While the polar head groups of one layer (outer leaflet) of
phospholipids face the exterior, those of the other layer (inner
leaflet) face the interior.
• A nonpolar or hydrophobic central region results where the
fatty acid tails of the two layers are in contact with each other

Asymmetry
• The fatty acid tails of all the phospholipids are
structurally very similar to each other
• The identity of an individual phospholipid molecule is
determined by the alcohol within its head group
• Some phospholipids are found on the outer leaflet
while others are more commonly seen on the inner
leaflet
• Asymmetry distribution across membrane
• Phospholipids, glycoproteins, cholesterol, glycoproteins,
peripheral membrane protein
• Maintenance of membrane asymmetry  for normal
cell function

Fluid Mosaic Model
• Flowing sea (~ mosaic)
• Ability of lipids to diffuse laterally within the plane of the membrane
• Many of the membrane proteins retain the ability to undergo lateral motion and are
likened to icebergs floating within the sea of lipids

Lipid Rafts
• = Specialized cholesterol-enriched
microdomains within cell membranes
• Fatty acid chains of phospholipids w/i the
rafts are extended and more tightly packed
• Phospholipids with straight acyl chains, including
glycosphingolipids, are found in lipid rafts
• Lipid rafts are described as floating within the
fluid created by the poorly ordered lipids of
the surrounding portions of the membrane
• Function:
• cholesterol transport, endocytosis, and signal
transduction

Lipids
• The most abundant type of macromolecule in most cell membranes
• Provide basic structure & the framework of the membrane
• Regulate membrane function
• 3 types:
• Phospholipids
• Cholesterol
• Glycolipids

Phospholipids
• Most abundant membrane lipids
• Polar, ionic compounds
• Amphipathic in nature
• Hydrophilic (polar) head
• Phosphate and alcohol attached to it
• Alcohol can be serine, ethanolamine, inositol, or choline
• Names of phospholipids then include phosphatidylserine,
phosphatidylethanolamine, phosphatidylinositol, and
phosphatidylcholine
• Hydrophobic tail
• Long, hydrocarbon (hydrogen + carbon) fatty acid tail

Cholesterol
• Dispersed throughout cell membranes 
intercalating between phospholipids
• Amphipathic molecule
• Polar hydroxyl group + hydrophobic steroid ring
• Attached hydrocarbon tail (parallel to those of
phospholipids)
• Fits into the spaces created by the kinks of the
unsaturated fatty acid tails
• Decreasing the ability of the fatty acids to undergo
motion
• Causing stiffening and strengthening of the membrane

Glycolipids
• = Lipids with attached carbohydrate (sugars)
• Lower concentration than phospholipids and cholesterol (in cell
membrane)
• The carbohydrate portion is always oriented toward the outside of the
cell, projecting into the environment
• Function:
• Help to form the carbohydrate coat observed on cells and are involved in cell-
to-cell interactions
• Source of blood group antigens
• Act as receptors for toxins including those from cholera and tetanus

Membrane Proteins
• Responsible for many biological functions of the membrane
• Transport of material into and out of cells
• Receptors for hormone or growth factors
• Types of proteins w/i plasma membrane vary depending on cell type
• A/w membrane in 1 of 3 main ways:
• Transmembrane proteins
• Lipid-anchored proteins
• Peripheral membrane proteins

3 Ways
Transmembrane proteins Lipid-anchored proteins Peripheral membrane proteins
• Embedded within the lipid bilayer
• Hydrophilic portions in contact with
the aqueous exterior environment &
cytosol
• Hydrophobic portions in contact with
the fatty acid tails of the
phospholipids
• Attached covalently to a portion of a
lipid without entering the core portion
of the bilayer of the membrane
• Integral membrane proteins (like TP)
• Removal will disrupt entire
membrane structure
• Located on the cytosolic side of the
membrane
• Only indirectly attached to the lipid of
the membrane; they bind to other
proteins that are attached to the
lipids.
• Iron channels or transport protein • G proteins which are named for their
ability to bind to guanosine
triphosphate (GTP) and participate in
cell signaling in response to certain
hormones
• Cytoskeletal proteins, such as those
involved in the spectrin membrane
skeleton of erythrocytes, are examples
of peripheral membrane proteins

MP
Function
Cell adhesion
molecules
• Proteins extend to the surface of cells  enable cell-to-cell contact
Ion channels
(transport
proteins)
• Enable molecule to enter & exit a cell
Ligand
receptors
• Enable cells to respond to hormones and other signalling molecules
Cytoskeletal
proteins
• Peripheral membrane protein attach tot eh membrane
• Regulate membrane shape and stabilize it structure

D e s c r i b e a n d c o m p a r e b e t w e e n
t h e s e t h r e e t y p e s o f m i c r o s c o p e s ,
c o m p o u n d l i g h t m i c r o s c o p e ,
t r a n s m i s s i o n a n d s c a n n i n g
e l e c t r o n m i c r o s c o p e

Describe the differences
between prokaryote and
eukaryote cells

Describe the structure
and function of these
organelles.

Nucleus
• All eukaryotic cells except mature erythrocytes (red blood cells) contain a nucleus
(plural = nuclei)
• Location where cell’s genomic DNA resides
• In cells that are not actively dividing, the DNA is contained within
chromosomes
• Every normal human cell contains 23 pairs of chromosomes within the
nucleus of every cell
• Nuclear envelope
• outermost structure, double-layered phospholipid membrane
• With nuclear pores to permit transfer of materials between the nucleus and
the cytosol
• Nucleoplasm
• Contain inside of nucleus
• The fluid in which the chromosomes are found
• Organized by the nuclear lamina, the protein scaffolding of the nucleoplasm
that is composed mainly of intermediate filaments
• Nuclear lamina
• Forms associations between the DNA and the inner nuclear membrane
• Nucelolus
• A prominent suborganelles within the nucleus
• Site of ribosome production

Endoplasmic Reticulum
• Appearing like a series of interconnected, flattened tubes,
• Often observed to surround the nucleus
• The outer layer of the nuclear envelope is actually contiguous with the
ER
• In muscle cells, this organelle is known as the sarcoplasmic reticulum
• The ER forms a maze of membrane-enclosed, interconnected spaces
that constitute the ER lumen, which sometimes expand into sacs or
cisternae
• Regions of ER where ribosomes are bound to the outer membrane are
called rough endoplasmic reticulum (rough ER or rER)
• Bound ribosomes and the associated ER are involved in the production
and modification of proteins that will be inserted into the plasma
membrane, function within lysosomes, Golgi complex, or ER, or else will
be secreted outside the cell
• Smooth endoplasmic reticulum (sER) refers to the regions of ER without
attached ribosomes
• Both rER and sER function in the glycosylation (addition of
carbohydrate) of proteins and in the synthesis of lipids

Golgi Complex
• Appears as flat, stacked, membranous
• 3 regions are described within the Golgi complex:
• Cis
• closest to the ER
• Medial & Trans
• near the plasma membrane
• Each region is responsible for performing distinct modifications
• Glycosylation (addition of carbohydrate)
• Phosphorylation (addition of phosphate)
• Proteolysis (enzyme-mediated breakdown of protein)
• To the newly synthesized proteins being processed and converted into
mature, functional proteins
• The trans Golgi network sorts and packages the newly synthesized and
modified proteins into distinct regions within the trans Golgi
• These regions bud off from the main body of the Golgi complex and form
structures called transport vesicles
• Movement of these new proteins toward their final cellular or extracellular
destination is facilitated in this manner.

Mitochondria
• Unique membranes to generate ATP
• Greatly increasing the energy yield from the breakdown of
carbohydrates and lipids
• Can self replicate, also contain their own DNA
• The very survival of individual cells depends on the integrity
of their mitochondria
• Programmed cell death or apoptosis occurs when pores are
formed in the mitochondrial membrane allowing for the
release of proteins that facilitate the apoptotic death process
• The unique structure of mitochondria is important in allowing
them to perform these necessary cellular functions

Mitochondria – Energy Production
• Double phospholipid bilayer membranes that form the outer
boundary of
• Inner mitochondrial membrane
• Forms folded structures called cristae
• Protrude into the mitochondrial lumen (space) known as the
mitochondrial matrix
• Protons (H+) are pumped out of the mitochondrial matrix, creating
an electrochemical gradient of protons
• The flow of protons back into the matrix drives the formation of ATP
from carbohydrates and lipids in the process of oxidative
phosphorylation
• The presence of mitochondria within a cell enhances the amount of
ATP produced from each glucose molecule that is broken down
• As evidenced by human red blood cells that lack mitochondria
• In red blood cells, only 2 ATP molecules are generated per glucose
molecule
• In contrast, in human cells with mitochondria, the yield of ATP is as high
as 32 per glucose molecule.

Mitochondria – Independent Units
within Eukaryotic Cells
• Mitochondria also contain DNA (mtDNA) and ribosomes for
the production of RNA and some mitochondrial proteins
• mtDNA is approximately 1% of total cellular DNA and exists in
a circular arrangement within the mitochondrial matrix
• Mutations or errors in some mitochondrial genes can result in
disease
• Most mitochondrial proteins, however, are encoded by the
genomic DNA of the cell’s nucleus
• Mitochondria self replicate or divide by fission, as do bacteria
• Mitochondria are actually believed to have arisen from
bacteria that were engulfed by ancestral eukaryotic cells.

M i t o c h o n d r i a – C e l l S u r v i v a l
• Survival of eukaryotic cells depends on intact mitochondria
• Mitochondrial involvement is important to ensure cell survival when it is
appropriate and also to facilitate programmed cell death when
necessary
• When the process of programmed cell death or apoptosis is stimulated
in a cell, proapoptotic proteins insert into the mitochondrial membrane,
forming pores
• A protein known as cytochrome c can then leave the intermembrane
space of the mitochondria through the pores, entering the cytosol
• Cytochrome c in the cytosol stimulates a cascade of biochemical
events resulting in apoptotic death of the cell

Lysosomes
• Membrane-enclosed organelles of various sizes that have an
acidic internal pH (pH 5)
• Formed from regions of the Golgi complex that pinch off when
proteins destined for the lysosome reach the trans Golgi
• Contain potent enzymes known collectively as acid
hydrolases
• These enzymes are synthesized on ribosomes bound to
the ER
• They function within the acidic environment of lysosomes
to hydrolyze or break down macromolecules (proteins,
nucleic acids, carbohydrates, and lipids)
• Lysosomes play a critical role in the normal turnover of
macromolecules that have reached the end of their
functional life

Lysosomes
• Nonfunctional macromolecules build up to toxic levels if they
are not degraded within lysosomes and properly recycled for
reuse within the cell
• This is exemplified by diseases known as lysosomal storage
diseases
• Such diseases are caused by defective acid hydrolases,
resulting in accumulation of substrates of the defective acid
hydrolases
• Most are fatal at an early age
• In infantile Tay Sachs disease, gangliosides accumulate in
the brain and death occurs by age
• In addition to degrading cellular macromolecules at the end of
their lifespan, lysosomal enzymes also degrade materials that
have been taken up by the cell through endocytosis or
phagocytosis

Peroxisomes
• Resemble lysosomes in size and in structure
• Have single membranes enclosing them and contain hydrolytic
enzymes
• However, they are formed from regions of the ER as opposed to
regions of the Golgi complex
• Enzymes that function in peroxisomes are synthesized on free
ribosomes and are not modified in the ER or Golgi complex
• Within peroxisomes, fatty acids and purines (AMP and GMP) are
broken down
• Hydrogen peroxide, a toxic by-product of many metabolic reactions,
is detoxified in peroxisomes
• Within liver cells (hepatocytes), peroxisomes participate in
cholesterol and bile acid synthesis
• Peroxisomes are also involved in the synthesis of myelin, the
substance that forms a protective sheath around many neurons

Compare between
passive transport and
active transport.

Stem Cells
• Unspecified or partially specified cells
which have the capacity to proliferate
or self-renew and differentiate into a
variety of cell types
• Exist in both:
• Early embryos
• Have an extensive capacity to differentiate
into all cell types of the organism
• Adult tissues
• Can differentiate into various cells within a
lineage but not outside that lineage

Describe totipotency,
pluripotency, unipotency
and multipotency
Specification potential of different stem cell types

Stem Cells Classification
Totipotency • The potential of a single cell to develop into a total organism
Pluripotency • A cell’s capacity to give rise to all cell types in the body but
not to the supporting structures (placenta, amnion, and
chorion, all of which are needed for the the development of
an organism)
Unipotency • A cell’s capacity to give rise to only one cell type
Multipotency • A cell’s capacity to give rise to a small number of different
cell types

Totipotent
• Generate all the cell types
in the organism
• Zygote or fertilized egg
• Blastomere  Blastocyst
• Produced by division of the
zygote after fertilization
• Capable of developing from a
single cell into a fully fertile
adult organism

Pluripotent Stem Cells
• The most primitive, undifferentiated cells in an embryo
• Embryonic stem cells
• Derived from the inner cell mass of the blastocyst
• Generate all the embryonic germ layers (i.e. endoderm, ectoderm and
mesoderm), but are unable to generate extra-embryonic tissues (i.e.
placenta)
• Plasticity
• = Ability to differentiate into a multiple cell types

Unipotent Stem Cells
• Adult stem cells
• Retain the ability to generate cells for the
tissue type to which they belong are
unipotent stem cells
• Have been identified for a number of
different tissue types
• Brain, bone marrow, peripheral blood, blood
vessels, skeletal muscle, skin, and liver

What is ECM? Describe
the composition of ECM.

Extracellular Matrix
• A large organised meshwork of
macromolecules in the extracellular space
• The macromolecules secreted by cells that live
within the tissue
• 3 macromolecules make up the ECM:
• Proteoglycans & Glycosaminoglycans (GAGs)
• Fibrous proteins (collagen & elastin)
• Adhesive proteins (fibronectin & laminin)
• Different from tissue to tissue
• Specialised to perform different functions
• Physical nature
• Cellular and extracellular compositions

Glycosaminoglycans (GAGs)
• AKA mucopolysaccharides
• Composed of repeated disaccharide chains
• N-acetylated amino sugar (N-acetylglucosamine / N-
acetylgalactosamine) + Acidic sugar
• Most are sulfated
• Organized in long, unbranched chains
• Contain multiple negative charges and are extended
in solution
• Most prevalent GAG = chondroitin sulfate
• Other GAGs include hyaluronic acid, keratin sulfate,
dermatan sulfate, heparin, and heparan sulfate.

Proteoglycans Characteristics
• Resilience of GAGs
• Net negatively charged surface of GAGs  repel each other
• Because of their net negative surface charges
• GAGs tend to slide past each other  producing the slippery consistency we associate
with mucous secretions
• Allow water floods into the matrix containing GAGs  creates swelling pressure (turgor)
• This pressure is balanced by the tension from collagen (fibrous protein of the
ECM), helping the ECM resist opposing forces of tissue compression

E.g. Knee Cartilage ECM
• Cartilage matrix lining the knee joint has large quantities
of GAGs and is also rich in collagen
• tough, resilient, and resistant to compression
• Cushion the bone of joints by the water balloon–like structure
of the hydrated GAGs in the cartilage
• When compressive forces are exerted on it, the water is
forced out and the GAGs occupy a smaller volume
• When the force of compression is released, water floods
back in, rehydrating the GAGs, much like a dried sponge
rapidly soaking up water.
• This change in hydration in the ECM is referred to as
resilience and is seen in cartilage as well as in synovial
fluid and the vitreous humor of the eye

Proteoglycans Structure
• With the exception of hyaluronic acid, GAGs are found
covalently attached to protein and form proteoglycan
monomers
• These monomers consist of a core protein with GAGs
extending out from it and remaining separated from each
other owing to charge repulsion
• In cartilage proteoglycans, the GAGs include chondroitin
sulfate and keratin sulfate
• The resulting proteoglycan is often described as having
a “bottle brush” or “fir tree” appearance

Proteoglycans Structure
• The individual GAG chains resemble needles on an
evergreen tree and the core protein, a branch
• The trunk of the tree is the hyaluronic acid, as
individual proteoglycan monomers then associate
with this large GAG, to form proteoglycan
aggregates
• The association occurs primarily through ionic
interactions between the core protein and the
hyaluronic acid and is stabilized by smaller, link
proteins

Fibrous Proteins
• Fibrous proteins are extended molecules that serve structural
functions in tissues
• They are composed of specific amino acids that combine into regular,
secondary structural elements
• Collagen and elastin are fibrous protein in the ECM that are
important components of CT as well as skin and blood vessel walls

Collagen
• The most abundant protein in the human body
• Forms tough protein fibers that are resistant to shearing forces
• Main type of protein in bone, tendon, and skin
• Bundled collagen in tendons imparts strength
• In bone, collagen fibers are oriented at an angle to other collagen fibers to
provide resistance to mechanical shear stress applied from any direction
• In the ECM, collagen is dispersed as a gel-like substance and provides support
and strength

Collagen
• Collagen is a family of proteins, with 28 distinct types
• However, over 90% of collagen in the human body is in collagen types I, II, III, and
IV.
• Together the collagens constitute 25% of total body protein mass
• Types I, II, and III are fibrillar collagens whose linear polymers of fibrils reflect the
packing together of individual collagen molecules
• Type IV (and also type VII) is a network-forming collagen that becomes a three-
dimensional mesh rather than distinct fibrils.

Collagen Structure
• Triple helix
• 3 helical polypeptide α chains of amino acids
 wind around one another forming a collagen
triple helical structure
• The various types of collagen have
different α chains, occurring in distinct
combinations
• Collagen type I = two type I α1 chains and one
type I α2 chain
• Collagen type II = three type II α1 chains

Collagen Structure
• Repeating units of -X-Gly-Y-
• The amino acid sequence of most of
the α chains can be represented as
repeating units of -X-Gly-Y-
• X = proline or lysine (hydroxyproline or
hydroxylysine),
• Gly = glycine
• Y = proline

Collagen Structure
• In the collagen triple helix, the small hydrogen
side chains of glycine residues (amino acids
within proteins) are placed toward the interior
of the helix in a space too small for any other
amino acid side chain
• Three α chains in this conformation can pack
together tightly
• Proline also facilitates the formation of the
helical conformation of each α chain because
it has a ring structure that causes “kinks” in the
pep- tide chain

C h a i n C o m p o s i t i o n s o f C o l l a g e n Ty p e s

Elastin
• The other major fibrous protein in the ECM
• Form elastic fibers  enable skin, arteries, and lungs to stretch and
recoil without tearing
• Rich in the amino acids glycine, alanine, proline, and lysine.
• Similar to collagen, elastin contains hydroxyproline, although only a
small amount
• No carbohydrate is found within the structure of elastin and therefore it
is not a glycoprotein

Elastin Synthesis
• Cells secrete the elastin precursor, tropoelastin, into the extracellular space.
• Tropoelastin then interacts with glycoprotein microfibrils including fibrillin,
which serve as a scaffolding onto which tropoelastin is deposited
• Side chains of some of the lysine amino acid residues within tropoelastin
polypeptides are modified to form allysine residues
• In the next step, the side chains of three allysine residues and the side
chain of one unaltered lysine residue from the same or neighbouring
tropoelastin polypeptide are joined covalently to form a desmosine cross-
link
• Thus, four individual polypeptide chains are covalently linked together

Elastin
• The structure of elastin is that of an
interconnected rubbery network that can impart
stretchiness to the tissue that contains it
• This structure resembles a collection of rubber
bands that have been knotted together, with the
knots being the desmosine cross-links
• Elastin monomers appear to lack an orderly
secondary protein structure because elastin can
adopt different conformations both when
relaxed and when stretched

Adhesive Proteins
• Proteins that join together and organize the ECM and
also link cells to the ECM
• Fibronectin and laminin
• Adhesive glycoproteins secreted by cells into the
extracellular space
• Principal adhesive in:
• Connective tissues = Fibronectin
• Epithelial tissues = Laminin
• Multifunctional protein
• Contain 3 different binding domains  link them to cell
surfaces and to other components of ECM (proteoglycans
& collagen)

Describe scurvy, osteogenesis
imper fecta, Ehler s - Danlos
syndr ome, Mar fan syndr ome and
α 1- antitr ypsin deficiency.

Scurvy
• Dietary deficiency in vitamin C  aberrant collagen production
• Vit C def  absence of hydroxylation of proline and lysine amino acid residues 
defective pro-α chains  cannot form a stable triple helix.
• These abnormal collagen pro-α chains are degraded within the lysosomes of the cell
• Less normal, functional collagen available to provide strength and stability
to tissues
• Blood vessels become fragile  bruising occurs
• Prolonged wound-healing is slowed
• Gingival haemorrhage and tooth loss occur

Osteogenesis Imperfecta
• A family of inherited collagen disorders,
• AKA “brittle bone disease”
• Caused by any one of several inherited mutations in a collagen gene  decreased
production of collagen or in abnormal collagen
• Eight forms
• Some forms have more severe signs and symptoms than others
• Most types have autosomal dominant inheritance patterns and affected persons inherit a mutant gene
from one parent who is also affected.
• OI Type I
• Most common
• Mostly mild signs and symptoms including bones that fracture easily, especially prior to puberty
• May also have spinal curvature and hearing loss
• OI Type II
• Lethal prior to or shortly after birth

Ehlers-Danlos Syndrome
• A group of relatively uncommon disorders that result from inherited
defects in the structure, production, or processing of fibrillar collagen
• 6 major types (categorized based on signs and symptoms)
• Most are inherited as autosomal dominant traits
• All six types include joint involvement
• Most also affect skin
• Some of the more prominent signs and symptoms include joints that extend
beyond the normal range of movement and skin that is especially stretchy or
fragile

Marfan Syndrome
• Mutation occurs in the gene that codes for the fibrillin-1 protein essential for
maintenance of elastin fibers
• Because elastin is found throughout the body and is particularly abundant in
the aorta, the ligaments, and in portions of the eye, these sites are most
affected in individuals with Marfan syndrome
• Many affected individuals have ocular abnormalities and myopia (near-
sightedness) and abnormalities in their aorta
• They also have long limbs and long digits, tall stature, scoliosis (side-to-side
or front-to-back spinal curvature) or kyphosis (curvature of the upper spine),
abnormal joint mobility, and hyperextensibility of hands, feet, elbows, and
knees

a1-Antitrypsin deficiency
• α1-antitrypsin
• AKA α1-protease inhibitor and α1-antiprotease
• Most important physiological inhibitor of neutrophil elastase
• In the lungs of all individuals, the alveoli are chronically exposed to low levels of neutrophil
elastase, released from activated neutrophils
• Deficient α1-antitrypsin
• Reduced ability to inhibit elastase in the lung
• Inappropriate destruction of the lung elastin (catalysed by the protease elastase)
• Affected individuals are predisposed to emphysema
• Because the lung tissue cannot regenerate, the destruction of the connective tissues of
alveolar walls is not repaired and disease results

What is cell adhesion?
Describe all cell
adhesion molecules

Cell Adhesion
• The process by which cells interact and attach to neighbouring cells
through specialised molecules of the cell surface
• Can occur either through
• Direct contact between cell surfaces – cell junctions
• Indirect interaction between cells to surrounding ECM – adhesive protein

Adhesion in Developing Tissues
• Many tissues, including most
epithelial tissues, develop from a
precursor, the founder cell that
divides to produce copies of itself.
• These newly produced cells remain
attached to the ECM and/or to other
cells owing to cell adhesion
• A growing tissue is able to form
because the member cells remain
attached and do not travel elsewhere

Cell Junction
• Class of cellular structures consisting of multiprotein
complexes that provide contact or adhesion between
neighbouring cells or between a cell and the ECM
• Also maintain the paracellular barrier or epithelia and
control paracellular transport
• Especially abundant in epithelial tissues
• Junction Types
• Tight
• Adherens
• Desmosome
• Gap
• Hemidesmosome

• Embedded within the plasma
membranes of cells
• Extend from cytoplasm through the
plasma membrane to the extracellular
space
• Bind specifically to their ligands
• Cell adhesion molecules on other cells
• Cell surface molecules
• ECM components
• Interactions between individual
adhesion molecules are important in
adhesion during development and
also mediate for cell migration
• 4 families of adhesion molecules
function in cell-cell adhesion:
• Calcium-dependent CAM
• Integrins
• Cadherins
• Selectins
• Calcium-independent CAM
• Immunoglobulin superfamily
Cell Adhesion Molecules

Cadherins
• Hold cells together  maintain the integrity of a tissue
• Contain:
• Extracellular domains
• Bind to a cadherin on another cell
• Intracellular domains
• Bind to linker proteins of catenin family
• Catenin bind to actin cytoskeleton (Cytoplasmic internal scaffolding)
• Therefore, 2 linked cells via cadherins  their actin cytoskeleton are indirectly linked
as well
• Mediate long-lasting adhesion
• Important in maintaining the tissue structure
• Notable in embryonic development
• Gastrulation for the formation of the mesoderm, endoderm, ectoderm

Selectins
• More transient cell-to-cell adhesions
• For example, selectins are particularly important in the
immune system in mediating white blood cell migration to
sites of inflammation
• Extravasation of WBD
• Selectins are named for their “lectin” or carbohydrate-
binding domain in the extracellular portion of their structure
• A selectin on one cell interacts with a carbohydrate-
containing ligand on another cell

Immunoglobulin Superfamily
• Share certain structural characteristics of immunoglobulins
(antibodies)
• Fine-tune and regulate cell-to-cell adhesions
• Some facilitate adhesion of leukocytes to endothelial cells
lining the blood vessels during injury and stress
• Ligands for this family of adhesion molecules include other
members of the immunoglobulin superfamily as well as
integrins

Integrins
• Mediate cell-to-cell & cell-to-ECM adhesions
• Ligands = Collagen, fibrinogen, fibronectin, vitronectin
• Members of this family of homologous transmembrane, heterodimeric
proteins bind to their ligands with relatively low affinity; multiple weak
adhesive interactions characterize integrin binding and function
• Heterodimeric = Consist of 2 transmembrane chains, α and β
• At least 19 α and 8 β chains are known at present
• Different α and β chains combine to give integrins with distinct binding
properties
• The β2-type subunit is expressed exclusively by leukocytes (white blood
cells)
• Integrins provide essential links between the ECM and the intracellular
signalling pathways – important in cell apoptosis, differentiation survival,
transcription

Extravasation of Cells
• Selectin – Rolling
• In this process, a selectin on the
leukocyte binds to its ligand, often a
member of the immunoglobulin
superfamily on the surface of an
endothelial cell
• “Rolling” of the leukocyte along the
endothelium of the blood vessel then
ensues

• Integrin Activation
• Activation of an integrin on the
same leukocyte occurs, in an
inside-out fashion, owing to
signalling set off by the selectin
interacting with its ligand
• Activated Integrin – Arrest
• The activated integrin can then
bind to its ligand on the
endothelium, causing a firm arrest
of the leukocyte

• Diapedesis
• Movement through the endothelial
layer, and extravasation, or entry of the
leukocyte into the tissue
• Understanding of these traditional
three steps of rolling, activation, and
firm binding has recently been
augmented and refined
• Slow rolling, adhesion strengthening,
intraluminal crawling, and paracellular
and transcellular migration are now
recognized as separate, additional
steps
• Atherosclerosis = Extravasation of
monocytes  engulf excess lipids
become foam cells  form plaque that
calcified  occlusion of BV

Explain some examples
of defects in adhesion
molecules
Cancer
Leukocyte adhesion deficiency
Pemphigus

Adhesion and Disease
• Normal expression and function of adhesion molecules are required to maintain
health and to defend against disease
• Interrupted cell adhesion trigger diseases
• E.g. impaired adhesion molecule expression on leukocytes & endothelium  defect trafficking
or movement of immune cells to the site of inflammation within a tissue
• Other reasons of interrupted cell adhesion:
• Infectious agents
• Disease processes
• Increased adhesion-molecule expression can contribute to inflammatory
conditions
• Asthma and rheumatoid arthritis

Cancer
• Most cancers originate from epithelial tissue
• E-cadherin is critically important in organizing the epithelium
• The function of E-cadherin is altered in most epithelial tumours.
• Studies have shown that this loss of E-cadherin–mediated cell-to-cell
adhesion occurs during tumour progression and is also required for
subsequent tumour spreading or metastasis.

Leukocyte Adhesion Deficiency
• Rare but significant immunodeficiency
• Inherited defect in the β2 subunit of integrins, which is normally
exclusively expressed on leukocytes
• Leukocytes have an impaired ability to traffic to the sites of infection
and recurrent bacterial infections result
• Persons with LAD generally do not survive beyond two years of age

Pemphigus
• Blister develop as a result of failed cell-to-cell adhesion
• Autoimmune condition characterized by disruption of cadherin-mediated cell adhesions
• 3 types (vary in severity)
• Pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus
• All forms are caused by autoantibodies that bind to the proteins in a subfamily of the cadherins =
desmogleins
• Antibody binding to desmogleins prevents their function in cell adhesion
• Therefore, adjacent epidermal cells are unable to adhere to each other and blisters develop
• (Pemphigoid is a related group of blistering conditions in which autoantibodies to proteins
of hemidesmosomes impair cell attachment to the underlying basal lamina)

Explain what happens when
ther e an incr ease of adhesion
molecule expr ession and
inflammation
Asthma
RA

Asthma
• Expression of more than the usual number of adhesion molecules per
cell can result in enhanced migration of cells to a region and can lead
to inappropriate inflammation
• ICAM-1, a member of the immunoglobulin superfamily that normally
facilitates adhesion between endothelial cells and leukocytes after
injury or stress, has been implicated in the pathogenesis of asthma
• Increased ICAM-1 expression is observed in the respiratory tract of
individuals with asthma
• This may permit an inappropriately large number of immune cells to
migrate there, stimulating chronic inflammation

Rheumatoid Arthritis
• Increased expression of adhesion molecules
• In this autoimmune disease, bone cells may have increased
expression of adhesion molecules
• In rheumatoid arthritis, synovial inflammation is associated with
increased leukocyte adhesion
• Selective involvement of the integrin LFA-1 and of ICAM-2 has been
demonstrated
• Inhibition of certain adhesion molecules is a potential therapy for RA

Using a diagram, explain
the signaling mechanism
of G protein signaling

G e n e r a t i o n o f s e c o n d m e s s e n g e r s i n r e s p o n s e t o
G α q a c t i v a t i o n o f p h o s p h o l i p a s e C

G Protein Signalling
• G proteins
• Intracellular signalling proteins
• Ability to bind to guanosine triphosphate (GTP)
• Also possess GTPase activity (hydrolyse GTP to GDP)
• Two categories:
• Heterotrimeric G proteins
• Ras superfamily of G proteins
• Often called “small G proteins” since they are monomers that
resemble one subunit of the heterotrimeric G proteins
• Receive their signals from catalytic receptors that have been activated
by their ligand
• The overall effects of Ras signalling often involve induction of cell
proliferation, cell differentiation, or vesicle transport

• Heterotrimeric G proteins
• 3 subunits, α, β, and γ
• Join together in inactive form
• GDP bound to α subunit
• Certain Gα subunits interact with certain enzymes
• Gαs stimulates adenylyl cyclase
• Gαi inhibit adenylyl cyclase

• Hormone / neurotransmitter (1st messenger) 
receptor linked with G protein  activate enzymes 
produce second messengers  activate
serine/threonine protein kinases (enzymes) 
phosphorylation of substrates (on serine and threonine
amino acid residues)
• Changes in phosphorylation status of target proteins,
many of which are enzymes, can alter their activity
• The overall result is the biological response of the cell
to the hormone or neurotransmitter
• The biological response is often the regulation of a
biochemical pathway or the expression of a gene

Receptors and Heterotrimeric G
Protein Signalling
• Many hormones and
neurotransmitters have receptors
on their target cells that are linked
to G proteins
• G protein-coupled receptors
• 7 membrane-spanning regions
• ~ 400 have been identified
• > 90% in brain

Signalling Mechanism
• In response to the receptor
binding to the G protein complex,
the Gα subunit of the G protein
releases GDP and binds GTP

• Active G protein
• α subunit dissociates from the β and γ subunits
• Interacts with an enzyme whose function is
regulated by the G protein
• Adenylyl cyclase
• Enzyme activated by Gs protein signalling
• Convert ATP to cyclic AMP (cAMP) and inorganic
phosphate (PPi)
• cAMP = second messenger in Gs signalling
• The type of G protein that is activated and the
second messenger it regulates depend on the
ligand, the type of receptor, and the type of
target cell

• When hormone is no longer present,
the receptor will revert to its resting
state
• GTP is hydrolysed to GDP (by the
GTPase of the G protein), the enzyme,
such as adenylyl cyclase, is
inactivated, and the α subunit will
reassociate with β and γ subunits to
stop the signalling process

W h a t a r e R a s G p r o t e i n s ? H o w i s
t h i s s i g n a l l i n g m e c h a n i s m
a c t i v a t e d ? W h a t h a p p e n s w h e n
t h e r e i s m u t a t i o n t o t h i s
s i g n a l l i n g ?

RAS G Proteins
• Rar sarcoma virus
• Homologous to α subunits of heterotrimeric G proteins
• All Ras protein family members belong to a class of protein called small
GTPase, and are involved in transmitting signals within cells (cellular signal
transduction)
• Ras is the prototypical member of the Ras superfamily of proteins, which
are all related in three-dimensional structure and regulate diverse cell
behaviours
• When Ras is ‘switched on’ by incoming signals, it subsequently switched on
other proteins, which ultimately run on genes involved in cell growth,
differentiation, and survival

R a s s i g n a l l i n g
v i a a c t i v a t i o n
o f a
c y t o p l a s m i c
s e r i n e /
t h r e o n i n e
c a s c a d e

Ras Mutations and Cell Pr olifer ation
• Mutations in Ras genes result in Ras proteins that cannot hydrolyse
GTP to GDP to inactivate the signalling process
• The Ras protein then remains in the active state without stimulation of
the receptor and continues to send signals to induce progression
through the cell cycle
• The result is excessive cell proliferation that can lead to malignancy

Explain the stages of cell
division and cell cycle
What is Hayflick’s limit?

Cell Division & Cell Cycle
• AKA proliferation
• Additional new cells required, for growth or replace those normally lost
• Somatic cells are generated by the division of existing cells in an
orderly sequence of events  duplicate contents  divide to produce
2 identical daughter cells
• Essential mechanism of eukaryotic reproduction

Cell Division & Cell Cycle
• For a cell to generate two daughter cells, complete
copies of all of the cell’s constituents must be made
• Genetic information contained within multiple
chromosomes must be duplicated; the cytoplasmic
organelles and cytoskeletal filaments must be copied
and shared between the two newly formed daughter
cells
• The cell cycle may be broadly divided into 3 distinct
stages:
• Interphase
• Mitosis
• Cytokinesis

Interphase
• Period between successive rounds of nuclear
division
• Distinguished by cellular growth and new
synthesis of DNA
• Resulting in a duplication of cellular materials
so that there are sufficient materials for 2
complete new daughter cells
• Further divided into 3 phases
• G1 phase
• S phase
• G2 phase

G1 and G0 phases
• Gap that follows mitosis and the next
round of DNA synthesis
• G1 = Growth phase + Preparation S phase
• RNA and protein synthesis also take place
• Organelles and intracellular structures are
duplicated and cell grows during this phase
• The length of G1 phase is the most
variable among cell types
• Very rapidly dividing cells (e.g. growing embryonic
cells) spend very little time in G1

S phase
• Synthesis of nuclear DNA (DNA replication)
• Each of the 46 chromosomes in a human cell is
copied to form a sister chromatid
• ATP-dependent unwinding of the chromatin structure by
DNA helicase exposes the binding sites for DNA
polymerase that will catalyse the synthesis of new DNA
in the 5′ to 3′ direction
• Multiple replication forks are activated on each
chromosome in order to ensure that the entire genome is
duplicated within the time span of S phase
• Upon completion of DNA synthesis, chromosome strands
are condensed into tightly coiled heterochromatin
• The time for completion of this process is relatively constant
among cell types. Actively cycling cells spend approximately 6
h in S phase

G2 phase
• The gap between the completion of S
phase and the start of mitosis
• Time of preparation for the nuclear
division of mitosis
• Safety gap  allows the cell to ensure
that DNA synthesis is complete before
proceeding to nuclear division in mitosis
• Also contained with the G2 is a
checkpoint where intracellular regulatory
molecules assess nuclear integrity

M Phase
• Nuclear Division
• Continuous process
• Divided into 5 phases based on progress
made to a specific point in the overall
nuclear division
• After completion of nuclear division,
cytokinesis occurs, involving cytoplasmic
division and resulting in the formation of
two separate daughter cells from the one
parent cell

Hayflick’s limit
• The number of times a normal somatic, differentiated human cell population will divide
before cell division stops
• Does not apply to stem cells
• Some tissues require continuous cell replacement, such as skin and gut epithelia and erythrocytes
• These cells derive from progenitor stem cells that do not exhibit Hayflick’s limit
• Other cells that are subject to Hayflick’s limit rarely divide, such as cells of the endocrine system, or
not at all, such as neurons, during adult life
• Each time a cell undergoes mitosis, the telomeres on the ends of each chromosome shorten
slightly and irreversibly
• Although telomerase, a complex of RNA and protein helps maintain and repair telomeres by adding
telomeric repeats, telomeric material is eventually lost  contributing to cellular senescence or aging
• Cell division will cease once telomeres shorten to a critical length

How is the assessment
of cell proliferation
carried out?

Assessment of Cell Cycle
• Important for evaluation of disease progression
• Equally important to both cell biology and drug-discovery research are
methods used to evaluate cell proliferation and the role of agents that
promote or retard the cell cycle
• Although there are a number of tools and methods to assess
proliferation, they can basically be divided into those used
• To analyse cell proliferation
• To assess the cell cycle

Assessment of Cell Proliferation
• DNA synthesis
• Dilution of a cytoplasmic probe

DNA synthesis
• Measurement of the synthesis of new
(nascent) DNA
• Using modified analogues of thymidine (one
of the nucleoside building blocks of DNA)
• Radioactive tritiated (3H) thymidine is added
into tissue culture medium in which cells are
grown
• Because thymidine is exclusively used for
DNA synthesis, cells that are actively
synthesizing DNA will incorporate 3H-
thymidine and the accumulated radioactivity
can be measured

Dilution of a Cytoplasmic Probe
• Cells are labelled with the carboxyfluorescein succinimidyl
ester (CFSE)
• CFSE  readily diffuse across plasma membranes and into
the cytoplasm
• There, intracellular esterases cleave the acetate groups of
CFSE making the compound both fluorescent and membrane
impermeant, thus trapping CFSE within the cell
• The succinimidyl ester groups of CFSE readily and irreversibly
bind to available amines (usually on lysine) on intracellular
cytoplasmic and membrane proteins
• As cells divide, their fluorescently labelled cytoplasmic
proteins are divided equally between the two daughter cells
• Each daughter cell has half the fluorescence of the previous
generation, which can be measured by flow cytometry

Cell Cycle Analysis
• The amount of DNA contained within a cell is cell cycle
dependent and ranges between 1n in G1 phase and 2n in
G2 and M phases
• Cell cycle distribution within a cell population can be
assessed by flow cytometry and is a clinically important
tool both in evaluating treatment therapies in lymphomas
and leukemias and as a research tool in evaluating
oncogene and tumor suppressor gene mechanisms
• Briefly, any one of a wide variety of nucleic acid–binding
fluorescent dyes may be used to label DNA
• Fluorescence is proportional to the DNA content of the cell
• Analysis of a flow cytometry histogram shows the
proportion of cells within the population in G1, S, and G2
phases of the cell cycle

What are the cell
cycle regulators?

Cell Cycle Regulators
• Control cell cycle progression
• The patterns of expression of these proteins and
enzymes depend upon the cell cycle phase
• Cell cycle mediators are categorized as cyclins or
as cyclin-dependent kinases (CDKs)
• Complexes of certain cyclins with specific CDKs
(cyclin-CDKs) possess enzymatic (kinase) activity
• Whenever necessary, cyclin-dependent kinase
inhibitors (CKI) can be recruited to inhibit cyclin-
CDK complexes

Cyclins
• The cyclins, categorized as cyclins D, E, A, or B, are a family
of cell cycle regulatory proteins
• Different cyclins are expressed to regulate specific phases of the cell
cycle
• Cyclin concentrations rise and fall throughout the cell cycle due
to its synthesis and degradation (via the proteosomal pathway
• Several categories of cyclins are known
• D-type cyclins (cyclins D1, D2, and D3) are G1 regulators critical for
progression through the restriction point, the point beyond which a
cell irrevocably proceeds through the remainder of the cell cycle
• S phase cyclins include type E cyclins and cyclin A
• Mitotic cyclins include cyclins B and A.

Cyclin-
dependent
Kinases
• Present in constant amount during cell cycle, but fluctuant enzyme
activities (depend on available cyclin concentrations for activation)
• Certain cyclins form complexes with certain CDKs to stimulate the kinase
activity of the CDK
• Only cyclin-CDK complex is an active kinase but its activity can be typically
further modulated by phosphorylation and other binding protein (p27)
• Involve in regulating transcription, mRNA processing, differentiation of
nerve cells

Explain tumour
suppressors and
checkpoints

Checkpoint Regulation
• Checkpoints placed at critical points in the cell
cycle monitor the completion of critical events
and, if necessary, delay the progression to the
next stage of the cell cycle
• One such checkpoint is the restriction point in
G1
• The cell depends on external stimuli from
growth factors to progress through the cell
cycle prior to the restriction point
• After that, the cell continues through the cell
cycle without the need for further stimulation

Tumour Suppressors & Checkpoints
• Tumour suppressor proteins
• To halt the cell cycle progression within G1 when the cell should not continue
past the restriction point
• Sometimes it is desirable for a cell to remain in G1 (or enter G0) when
continued growth is not needed or undesirable or when DNA is damaged
• Mutated versions of tumour suppressor genes may encode proteins
that permit cell cycle progression at inappropriate times
• Cancer cells often show mutations of tumour suppressor genes

Checkpoint Regulation
• It is important that nuclear synthesis of
DNA not begin until all the appropriate
cellular growth has occurred during G1
• Therefore, there are key regulators that
ensure that G1 is completed prior to the
start of S phase

Retinoblastoma (RB) Protein
• Tumour suppressor proteins (halt a cell in the G1 phase)
• RB gene mutation
• Found in an inherited eye malignancy known as hereditary RB
• The mutant gene encodes an RB protein unable to halt the cell cycle in G1
allowing unregulated progression through the remainder of the cell cycle

RB activation of tr anscr iption of
S phase genes
• In resting cells, the RB protein
contains few phosphorylated
amino acid residues
• In this state, RB prevents entry
into S phase by binding to
transcription factor E2F and its
binding partner DP1/2 which are
critical for the G1/S transition
• Therefore, RB normally prevents
progression out of early G1 and
into S phase in a resting cell

RB activation of tr anscr iption of
S phase genes
• In actively cycling cells, RB is progressively hyperphosphorylated as a consequence of
growth factor stimulation and signalling via the MAP kinase cascade
• Subsequently, cyclin D-CDK4/6 complexes are activated and they phosphorylate RB
• Further phosphorylation of RB by cyclin E-CDK2 allows the cell to move out of G1
• Hyperphosphorylated RB can no longer inhibit transcription factor E2F binding to DNA
• Therefore, E2F is able to bind to DNA and activate genes whose products are important
for S phase
• Examples of E2F-regulated genes include thymidine kinase and DNA polymerase
• Both of which are involved in the synthesis of DNA

What happens when
DNA damage occurs?

D N A D a m a g e & C e l l C y c l e C h e c k p o i n t s
• The usual response to DNA damage is to halt the cell cycle in G1, until DNA
repair can be accomplished
• However, depending on the type of DNA damage, different cell cycle
regulatory systems may be utilized
• Types of DNA damage:
• Endogenous
• Replication errors
• Exogenous
• Chemical exposure
• Oxidative insults
• Cellular metabolism

D N A d a m a g e -
i n d u c e d r e s p o n s e s
• Tumour suppressors ATM (ataxia
telangiectasia, mutated) & ATR (ATM
and Rad3 related) respond to distinct
types of DNA damage
• ATM
• Primary mediator of the response
to double-strand DNA breaks
• Type of damage induced by
ionizing radiation
• ATR
• Mediating UV-induced DNA
damage
• But it has a secondary role in the
response to double-strand DNA
breaks.

D N A d a m a g e -
i n d u c e d r e s p o n s e s
• The S phase checkpoint monitors
cell cycle progression and slows
the rate of DNA synthesis should
DNA damage occur to an S phase
cell
• BRCA1, the protein product of the
breast cancer susceptibility gene 1,
plays a role in the repair of double-
strand DNA breaks as part of a large
complex
• The mechanistic details and other
proteins involved remain to be
elucidated

DNA Repair Systems
• DNA repair
• Necessary not only because cells are continuously exposed to environmental
mutagens
• But also because thousands of mutations would otherwise occur spontaneously
in every cell each day during DNA replication
• In most of these cases, the cells use the undamaged strand of DNA
as a template to correct the mistakes in DNA
• When both strands are damaged, the cell resorts to the use of the sister
chromatid (the second copy of DNA present in diploid cells) or to an error-prone
recovery mechanism
• All types of repair mechanisms are made up of enzymes that follow
a general scheme of recognition, removal, repair, and relegation
• However, depending on the type of damage, different enzymes are
employed

Explain the basis of some
chemotherapeutic agents
specific to cell cycle

C h e m o t h e r a p e u t i c A g e n t s a n d C e l l C y c l e
• Both normal and tumour cells utilize the same cell cycle
• Normal and neoplastic (cancerous) tissue may differ in
the total number of cells in active phases of the cell cycle
• Some chemotherapeutic agents are effective only in
actively cycling cells
• These therapies are considered to be cell cycle–specific
agents and are generally used for tumours with a high
percentage of dividing cells
• Normal, actively cycling cells are also damaged by such
therapies
• When tumours have a low percentage of dividing cells,
then cell cycle–nonspecific agents can be used
therapeutically

Antimetabolites
• Compounds structurally related to normal
cellular components are called
antimetabolites
• Exert their toxic effects on cells in the S
phase of the cell cycle
• MOA
• Inhibition of synthesis of purine or pyrimidine
nucleotide precursors
• Compete with nucleotides in DNA and RNA
synthesis
• Methotrexate and 5-fluorouracil

Anticancer Antibiotics
• Bleomycin
• Cause cells to accumulate in the G2 phase of the cell
cycle
• Other anticancer antibiotics are not specific to any
particular cell cycle phase but do impact actively
cycling cells more than resting cells
• Their mechanism of action involves interacting with
DNA and disrupting DNA function
• Some alkylating agents and nitrosoureas are cell
cycle–nonspecific drugs
• These agents are often used to treat solid tumours
with low growth fractions

Mitotic Spindle Poisons
• Inhibit M phase cells
• Specifically during metaphase
• MOA
• Binding to tubulin + disrupting the spindle
apparatus of the microtubules required for
chromosome segregation
• Often used to treat high growth fraction
cancers (leukaemia)
• E.g. Vincristine, Vinblastine & Taxol

Necrosis
• A passive, pathological process induced by acute injury
or disease.
• A group of cells in a localized region of a tissue generally
undergo necrosis at the same time after experiencing an insult.
• Cells that die by necrosis increase in volume and lyse
(burst), releasing their intracellular contents.
• Mitochondria and other intracellular components are
released, often inducing a potentially damaging
inflammatory response.
• The necrotic process is completed within several days.

Apoptosis
• Cells deprived of survival factors activate an
intracellular suicide program and die by a process of
programmed cell death
• The requirement of a cell to receive signals for survival helps to
ensure that cells continue to live only when and where they are
needed

Apoptosis
• Cells undergoing apoptosis shrink in size but do not
lyse
• Their plasma membrane remains intact but portions of
the membrane eventually bud off, or bleb, and lose their
asymmetry and ability to attach to neighbouring cells in
a tissue
• The membrane phospholipid phosphatidylserine, which is
normally present on the inner membrane leaflet oriented
toward the cytosol, inverts and becomes exposed on the cell’s
surface

Cellular changes
during apoptosis
• In an active, ATP-requiring process, mitochondria
of apoptotic cells release cytochrome c but remain
within the membrane blebs
• Chromatin of apoptotic cells segments and
condenses

Apoptotic cell removal
via phagocytosis
• Apoptotic cells are engulfed by phagocytic cells,
macrophages and dendritic cells, which bind to
the phosphatidylserine on the membrane surface
• A macrophage internalizes and then degrades an
apoptotic cell, reducing the risk of inflammation
from the cell death
• Phagocytic cells also release cytokines including
interleukin-10 (IL-10) and transforming growth
factor-β (TGF-β) that inhibit inflammation

Apoptosis
• Therefore, there is no extensive damage done to
neighbouring cells in a tissue when a nearby resident
cell undergoes apoptosis
• Apoptosis is completed within a few hours

What is the function of
apoptosis?
Elimination of damaged cells
Development
Homeostasis

Elimination of
damaged cells
• Damaged (beyond repair) cells from:
• Viral infection
• Experiencing starvation
• Effects of ionizing radiation or toxins
• Actions of tumour suppressor protein p53 (a
product of the p53 gene) halt the cell cycle and
stimulate apoptosis

Elimination of
damaged cells
• The normal (wild type) p53 binds to a p53-responsive
element within the gene promoter of the proapoptotic
protein BAX  triggering programmed cell death
• But, mutant forms of p53 can neither halt the cell cycle
nor initiate apoptosis
• Therefore, abnormal cells expressing mutant p53 can
continue to divide  fail to undergo apoptosis
• Despite the fact that their survival damages the organism

Elimination of
damaged cells
• Removal of individual cells by apoptosis saves
nutrients needed by other cells
• Halt the spread of a viral infection to other cells

Development
• Embryo development
• Removal of excessive number of cells during
extensive cell division and differentiation
• For normal development to proceed and for normal
function to occur
• Selective apoptosis “sculpts” the developing tissue
• E.g., apoptotic death of cells between developing digits
must occur for formation of individual fingers and toes
• Incomplete apoptosis can result in abnormal structures

Homeostasis
• Keep number of cells in relatively constant
(balance between cell division – cell death)
• If the equilibrium is disturbed, then abnormal
growth and tumours or abnormal cell loss can
result

Sonic hedgehog (Shh) & Apoptosis
• Sonic hedgehog = Signalling pathway
• Sends an antiapoptotic signal  allow for cell survival
• Failure to receive the signal results in apoptosis
• Impaired Shh system
• Send inappropriate antiapoptotic signal
• Allow damaged cells to escape death
• Potential for development of malignancy

Explain the mechanism
of apoptosis with a
diagram

Favaloro, B., Allocati, N., Graziano, V., Di Ilio, C., & De Laurenzi, V. (2012). Role of apoptosis in disease. Aging (Albany NY), 4(5), 330.

Phases of Apoptosis
• Triggering phase (ligation of “dedicat3ed death receptor)
• Signalling phase (protein kinase cascades – MAPK family)
• Execution phase (activation of caspases and nucleases)
• Burial phase (phagocytosis of dying cells by neighbouring cells

Cellular Apoptosis via
Apoptosome Formation
• Irreparable DNA damage
• Bax protein insert into mitochondria membrane
• Cytochrome c exit mitochondria
• Cytoplasmic cytochrome c activates Apaf-1 adaptor
protein  activates caspase 9 (caspase proteolytic
cascade)
• Cleave & destroy cellular protein & DNA

Death-receptor Initiation
of Apoptosis
• Death receptors
• Belonging to the tumour necrosis factor
receptor superfamily (TNFRSF)
• Expressed on many cell types, especially in
the immune system
• Consists of death domain
• In cytoplasmic region
• Enables the receptors to initiate cytotoxic signals
when engaged by adaptor molecules (cognate
ligands)

Death-receptor Initiation
of Apoptosis
• Adaptor molecules (ligands)
• Examples:
• FADD (Fas-associated death domain)
• TRADD (TNFR-associated protein)
• Contain correspond death domains
• Interact with the death receptors
• Activation of activation of intracellular cysteine
proteases (caspase 8 or 10)
• Without directly involving the mitochondrial death pathway
• Transmit the apoptotic signal to the death machinery

E.g. Fas ligand-induced Apoptosis
• Adaptor molecule = Fas
ligand (FasL, aka CD 178)
• Expressed on T-cytotoxic cells
(membrane-anchored)
• Causes trimerization of Fas
death receptor  formed
death receptor trimer
• Death receptor = Fas death
receptor
• On host cells surface infected
with virus
• Bind by FasL on T-cell

• Clustering of receptors’
death domain
• Receptor DD recruit
cytosolic adaptor protein
FADD
• By binding to FADD death
domain

• FADD
• Contains death effector
domain (DED)
• That binds to an analogous
domain repeated in tandem
within procaspase 8 (inactive
or zymogen form of caspase
8)
• Formation of death-inducing
signalling complex (DISC)
• Fas receptor + FADD +
Caspase 8

• Caspase 8
• Activated itself from
procaspase 8
• Activates downstream
caspases  commits the cells
to apoptosis
• Apoptosis triggered by FasL-
Fas (CD178:CD95)
• Plays a fundamental role in
the regulation of the immune
system

Explain apoptosis in
cancer, autoimmune
conditions and
neurodegenerative illnesses

Disrupted balance of pro -apoptotic
and anti-apoptotic proteins
• Reduced or resistance to apoptosis
• Disrupted balance of pro-apoptotic and anti-apoptotic proteins
• Reduced caspase function
• Impaired death receptor signalling
Wong RS. Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin Cancer Res. 2011 Sep 26;30(1):87. doi: 10.1186/1756-9966-30-87. PMID: 21943236; PMCID: PMC3197541.

Bcl-2 Family of Proteins
• Bcl-2 (B-cell lymphoma 2) encoded in humans by BCL2 gene
• Founding member of Bcl-2 family (first apoptosis regulator identified)
• Regulatory proteins that regulate cell death by either inhibiting (anti-apoptotic) or inducing (pro-apoptotic) apoptosis
• All the Bcl-2 members are located on the outer mitochondrial membrane
• Dimers responsible for membrane permeability
• Consists of pro-apoptotic and anti-apoptotic proteins
• Antiapoptotic (prosurvival) – inhibit apoptosis
• Bcl-2
• Bcl-xL (B-cell lymphoma extra large)
• Proapoptotic (prodeath) – initiate apoptosis
• Bak (Bcl-2 homologous antagonist killer)
• Bax (Bcl-2-associated protein)
• In apoptotic cells, proapoptotic proteins > antiapoptotic
• In carcinogenesis, antiapoptotic proteins > proapoptotic

B c l - 2 F a m i l y M e m b e r s
i n A p o p t o s i s
• The Bcl-2 family proteins consists of
members that either promote or inhibit
apoptosis
• Control apoptosis by governing
mithocondrial outer membrane
permeabilization (MOMP)
• Key step in the intrinsic pathway of
apoptosis
• Result in diffusion of proteins (cytochrome
c) from the space between the inner and
outer mitochondrial membranes into the
cytosol
• Result in apoptosis by activation of
caspase cascade

p53 (Tumour Protein 53)
• One of the best known tumour suppressor proteins
• Encoded by tumour suppressor gene TP53 located at chromosome 17
• Named after its molecular weight (53kDa)
• Involve in induction of apoptosis
• Key player in cell cycle regulation, development, differentiation, gene
amplification, DNA recombination, chromosomal segregation and cellular
senescence
• “Guardian of genome”
• P53 mutation gain oncogenic function

Inhibitor of Apoptosis Proteins
(IAPs)
• Group of structurally and functionally similar proteins that regulate
apoptosis, cytokinesis, and signal transduction
• Characterised by presence of baculovirus IAP repeat (BIR) domain
• Endogenous inhibitors of caspases
• Binding to active caspase by their BIR domain
• Promoting degradation of active caspases or keeping the caspases away from
their substrates

Reduced Caspase Activity
• Caspases important in initiation and execution of apoptosis
• Impairment in caspase function may lead to decreased in apoptosis
and carcinogenesis
• More than one caspase can be downregulated, contributing to tumour
cell growth and development

Caspase Family of Proteases
• Proteases = enzyme whose substrate are proteins
• Major effectors of apoptotic cell death
• Members of cysteine protease class
• Named after a cysteine amino acid residue present within the catalytic site of
the enzyme molecule
• Synthesized in inactive zymogen or proenzyme forms and are
activated to become functional proteases when needed
• This posttranslational modification ensures that the enzymes can be activated
rapidly when required

Classification of Caspases
(based on function)
• 11 members have been identified in
humans
• Non-apoptotic caspase:
• Caspase 1  cytokine maturation
• Caspases 4 and 5  involved in inflammation
• Caspase 14  skin development
• Apoptotic caspase:
• Initiator caspase
• Effector caspase

Initiator Caspase
• Caspases 2, 8, 9, 10
• Possess characteristic regions or domains
• Caspase recruitment domains (CARD) in caspases 2 and 9
• Death effector domain (DED) in caspases 8 and 10
• CARD & DED interact with molecules that regulate their activity
• Initiator caspases cleave inactive proenzyme forms of effector
caspases, resulting in their activation

Effector Caspase
• Caspases 3, 6, 7
• “Executioner caspases”
• Proteolytically cleave protein substrates with the cell
• Causing the apoptotic demise of the cell

Caspase Cascade
• Sequential proteolytic activation of one caspase after another in an
orderly fashion during the initiation of apoptosis
• Caspase inhibitors regulate the process
• The cascade can be activated by various stimuli, including the
apoptosome, death receptors, and granzyme B released by cytotoxic
T cells
• Apoptosome  initiator caspases 9
• Death receptors  initiator caspases 8 and 10
• Granzyme B  effector caspases 3 and 7

Targets of Caspases
• Nuclear & cytoplasmic proteins.
• Nuclear lamins, structural fibrous proteins in the nucleus, are targets of caspases.
• Additionally, DNA fragmentation factor 45/inhibitor of caspase-activated
DNAse is cleaved, allowing caspase-activated DNAse to enter the
nucleus and fragment DNA, causing the characteristic laddering pattern
of DNA in apoptotic cells (Figure 23.11).
• The DNA is cleaved by an endonuclease into fragments that are
multiples of the same size, corresponding to the length of the
nucleosome coil, for example, 2, 4, 6, 8, etc.
• A distinctive 180-bp ladder is seen in the DNA of cells undergoing
apoptosis.
• Poly ADP ribose polymerase is also known to be proteolytically cleaved
by caspases during the apoptotic process, as is Bid, a member of the
Bcl-2 family.

Impaired Death Receptor Signalling
• Several abnormalities in the death signalling pathway that can lead to
evasion of the extrinsic pathway of apoptosis have been identified:
• Down-regulation of receptor
• Impairment of receptor function (any defects)
• Reduced level in the death signals
• Cause chemotherapy-resistance cancer
• Reduced expression of CD 95 – treatment-resistant leukaemia or
neuroblastoma cells

Targeting Apoptosis in Cancer
Treatment
• Therapy to restore the apoptotic signalling pathway towards normality
• Depends on / target the apoptotic defects
• To eliminate cancer cells
• Treatment strategies:
• Targeting the Bcl-2 family of proteins
• Targeting p53
• Targeting IAPS

Apoptosis and Autoimmune Disease
• A common feature of autoimmune diseases is altered tolerance to self
antigens and generation of autoantibodies
• Immune homeostasis and maintenance of immune tolerance are
strongly dependent on apoptosis, moreover defective clearance of
dying cells results in persistence of autoantigens
• Therefore, autoimmune disease can arise from both:
• Defective clearance of autoreactive cells
• Delayed elimination of autoantigens

• In addition increased apoptosis as a consequence of viral infections,
gamma irradiation or other stressing conditions may contribute to disease
onset
• More recently it has been suggested that upon apoptosis and/or secondary
necrosis autoantigens are cleaved and modified exposing novel epitopes
that are recognized by the immune system, again altered or delayed
clearance as well as prolonged exposure to apoptotic inducing stimuli would
result in autoimmune response
• Moreover formation of immune complexes would result in secretion of pro-
inflammatory cytokines such as IL-8, Il-1β, TNFβ and IFN-α resulting in
chronic inflammation and organ damage

• Autoimmune thyroid diseases
• Fas and FasL expression
• SLE
• RA

Autoimmune
thyroid diseases
• FasL expression on thyrocytes
• It was initially believed that FasL
expression was restricted to
activated cells
• But can also expressed on normal
thyrocytes  induce apoptosis of
infiltrated, activated T cells
• Protect normal thyrocytes against
attack by T cells
• Immunopreviledged site
Eguchi, K. (2001). Apoptosis in autoimmune diseases. Internal medicine, 40(4), 275-284.

Hashimoto
Thyroiditis
• Massive infiltration of lymphoid
cells  produce cytokine (IL-1B)
 induced Fas expression on
thyrocytes
• + Expression of FasL on
thyrocytes
• Result in suicide or fratricide
among neighbouring thyrocytes
• Hypothyroidism

Grave Disease
• Abnormal proliferation of thyrocytes > Apoptosis
• Lead to thyroid hyperplasia (Goiter)
• Increased production ofIL-1B by infiltrating mononuclear cells
• Stimulate thyrocytes show reduced cytotoxic activity toward activated T cells
• Reduce resistance to Fas-mediated apoptosis & lose their cytotoxic activity
against activated T cells
• Abolishing the immunepreviledged status of thyroid gland
• Accumulation of activated T cells in thyroid tissue

Grave Disease

Apoptosis in SLE
• A common feature of autoimmune diseases such as SLE, systemic
sclerosis, Sjogren syndrome and mixed connective tissue disease is
the breakdown of tolerance of self antigens
• A consequence of which is the production of antibodies reactive with multiple
self proteins
• Repeated or persistent exposure to stimulus  sustained apoptosis 
continuous source of autoantigens  modified (caspase-mediated
cleavage of different cadres of autoantigens)  drive T and B cells 
development o autoantibodies  exert pathologic effects

Apoptosis in Rheumatoid Arthritis
• Pronounced hyperplasia of synovial tissue
• Infiltrated cells in synovial tissues
• Periarticular Osteoporosis

Pronounced hyperplasia of synovial
tissue
• Interaction between Fas antigen or synovial cells + FasL on activated
T cells  cause apoptosis of synovial cells
• Induce regression of proliferation of synovium which can be seen in
patients with RA
• However, the function of Fas/FasL system seems to be incapable of
eliminating the cells in the proliferating RA synovium
• Bcl-2 is highly expressed on synovial fibroblasts in the synovial lining
and the sublining region from RA

Pronounced hyperplasia of synovial
tissue
• Various cytokines such as IL-1B, Various cytokines such as IL-lp,
platelet-derived growth factor (PDGF), basic fibroblast growth
factor (bFGF), transforming growth factor B(TGF-B) and TNF-a
which are present in synovial tissues from RA patients, have been
shown to stimulate the proliferation of human synovial cells
• Stimulation of TGF-B becomes markedly resistant to Fas-mediated and
proteasome inhibitor-mediated apoptosis

Infiltrated cells in synovial tissues
• The inflammatory infiltrate in RA comprises T, B cells, macrophages
and neutrophils
• Despite the increased expression of Fas and FasL on infiltrating T
cells, in situ observations of synovial lymphoid aggregates suggest low
levels of apoptosis
• This may due to:
• High expression of Bcl-2
• Production of an antiapoptotic factor by stromal cells
• Cell-to-cell interaction between lymphocytes and synovial cells

Periarticular Osteoporosis
• Periarticular osteoporosis is a clinical common features in RA
• Osteoblast apoptosis
• Evidence suggest that activated T cells in synovium or synovial fluid in RA patients express
membranous FasL and produce soluble FasL  induce apoptosis of osteoblasts
• Osteoclastogenesis
• Excess differentiation & activation of osteoclast in RA
• Role of NF-kB ligand (RANKL) and RANK
• RANKL expressed on osteoblast/stromal lineage cells
• RANK (receptor) expressed on osteoclast lineage cells
• Binding of RANKL to RANK induces differentiation, activation and survival of osteoclasts
• Cytokines such as (IL-1B, -6, -11, -17 and TNF-a which are abundant in synovial tissues from RA 
increase the expression of RANKL with a decrease in OPG expression on osteoblasts/stromal cells 
differentiation and activation of osteoclasts

Apoptosis in Neurodegenerative
Diseases
• From physiological point of view apoptosis plays a key role in central
nervous development, while in adult brain it is involved in the
pathogenesis of a number of diseases including neurodegenerative
• Alzheimer’s disease
• Parkinson’s disease

Alzheimer ’s disease (AD)
• Progressive neurodegenerative disorder
• Neuronal apoptosis plays an important role in AD pathogenesis and
caspases seem to be involved also in some of the upstream
pathological events
• Exposure of cultured hippocampal neurons to β results in caspase 3
activation and apoptosis
• Localised apoptosis may contribute to early neurite and synapse loss, leading
to the initial cognitive decline

Parkinson’s disease (PD)
• Second most common chronic neurodegenerative disorder after AD
• A/w movement disorders, tremors, and rigidity
• Characterised by a specific loss of dopaminergic neurons of the
substantia nigra
• This degeneration leads to the formation of fibrillar cytoplasmic
inclusions known as Lewy bodies
• A preponderant role of the aberrant activation of intrinsic and extrinsic
apoptotic pathways in PD pathogenesis has been suggested

Schizophrenia
• Chronic neurodegenerative illness
• Characterized by delusions, hallucinations, and changes in emotional
state
• Although the mechanisms underlying these deficits are largely
unknown, recent postmortem data implicate a role for altered neuronal
apoptosis
• Apoptotic regulatory proteins and DNA fragmentation patterns appear
to be altered in several cortical regions in individuals with
schizophrenia

HIV-associated Dementia (HAD)
• Many individual infected with HIV virus develop this syndrome of
neurologic deterioration
• Appears to be associated with active caspase 3 in the affected brain
regions, leading to speculation that pharmacologic interventions aimed
at the caspase pathway may be beneficial

Explain four laboratory
methods to assess
apoptosis
DNA laddering
TUNEL
Annexin 5
Flow cytometry

DNA Laddering
• Oldest technique available to detect that
apoptosis
• Since the genomic DNA of apoptotic cells is
degraded into approximately 180 base pair
fragments, a characteristic laddering
appearance is revealed on agarose gel
electrophoresis

TUNEL
• Terminal Uridine Deoxynucleotidyl Transferase nick end
labelling
• TUNEL is a method for detecting apoptotic
DNA fragmentation
• Widely used to identify and quantify apoptotic cells, or to
detect excessive DNA breakage in individual cells
• The assay relies on the use of terminal deoxynucleotidyl
transferase (TdT), an enzyme that catalyses attachment of
deoxynucleotides, tagged with a fluorochrome or another
marker, to 3'-hydroxyl termini of DNA double strand breaks
• It may also label cells having DNA damage by other
means than in the course of apoptosis

Annexin 5
• Annexins = family of proteins that bind to
phospholipids in cell membranes
• Annexin 5 binds to phosphatidylserine
• In healthy cells  present on the inner membrane
leaflet
• Apoptotic cells  flip-flops to the outer membrane
leaflet
• A labelled antibody to Annexin 5 can be used
to detect cells displaying phosphatidylserine
on their outer leaflet
• Indicating that they have initiated the apoptotic
process

Flow Cytometry
• This procedure can be used to measure
cell size and granularity of cells within a
population, both of which differ in
apoptotic and normal cells
• Because apoptotic cells shrink in size,
the forward angle light scatter will reveal
an apoptotic population of less intensity
compared with normal cells
• Granularity of apoptotic cells is increased
compared with that of normal cells, as
indicated by side scatter

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