CELLULAR BASIS OF MEDICINE – COURSE 4

Course 4 notes

Molecular Biology
Cell cycle – lifecycle of a cell. It consists of:
• Interphase, which is quite long. It consists of:
o G1 phase – the cell grows in its size, proteins and extra organelles are
synthesized.
o S phase – DNA replication and centrosome duplication
o G2 phase – preparation for mitosis. Microtubules are being assembled
• M phase – includes mitosis and cytokinesis (comparing to interphase it is much
shorter). It consists of:
o Prophase – centrosomes are moving to the poles of the cell, while nuclear
envelope disintegrates
(centrosomes are parts of the mitotic spindle where centrioles are located inside of the
pericentriolar matrix. Centrosome contains gamma tubulin and nuclear mitotic apparatus
which contributes to reorganization and stabilization of microtubules by interacting with
dynein and by assembling the mitotic spindle. The mitotic spindle itself consists of
centrosomes, microtubules and kinetochore.
o Prometaphase – centrosomes are at the edges of the cell, by that time
nuclear envelope is disintegrated completely
o Metaphase – condensed chromosomes are in the equatorial position and
microtubules attach to the chromosomes by kinetochores
o Anaphase – chromosomes are dragged by microtubules in the opposite
directions to the pole. There are two stages: Anaphase A – chromosomes are
pulled to the poles due to kinetochore shortening, Anaphase B – the poles
are moving apart, therefore, separating chromosomes even further. Also
cytokinesis begins around this time.
o Telophase – chromosomes are fully separated by centrosomes and the new
nuclear membranes are formed. Cytokinesis ends at the end of telophase.
There are some cells which are in the G0 phase, those are called resting cells. Those cells are
usually permanent cells of our body such as neurons, cardiac muscle cells, cells of retina of
the eye and non-proliferating stem cells.
Other cells can be found in G0 phase as well, but they can move back into the division by
switching to the G1 phase. They can move back with the help of specific proteins, such as
Myc.

Regulation of the cell cycle

Primary response genes – code for the transcription factors that regulate secondary genes
• C-myc (key gene for activation)
• C-fos & c-jun – heterodimer – (AP-1 transcription factor)
Secondary response genes – code for the effector molecules for cell cycle execution
• CDK genes (progression of the cell cycle (catalytic subunit)
• Cyclin – binding the cyclin is necessary for the CDK activation (regulatory subunit)
• C-myb
Cyclin together with CDK form cyclin/CDK complex. In order for them to be activated one
site should be phosphorylated, whereas two sides should be dephosphorylated. When both

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of the requirements are fulfilled, the complex is active. This active complex then
phosphorylates relevant proteins therefore proceeding to the progression of the cell cycle
(transition to S phase or M phase).
When cyclin is degraded, the complex becomes inactive again. There are different types of
complexes facilitating specific transitions:
• Cyclin E/CDK2 – regulates G1 -> S transition
• Cyclin A/CDK1 – regulates S -> G2 transition
• Cyclin B/CDK1 – regulates G2 -> M transition
There are also specific inhibitors for the certain cyclin/CDK complexes which are binding to
them
• P21 family – p21
• INK4 family – p15, p16
The Rb protein (the Rb protein family includes p107, p130, pRb) is the main regulatory
protein in the cell cycle. It blocks the potential replication of damaged DNA. Also, its duties
include inhibition of activation (no transition from G0 to G1) and inhibition of progression
(no transition from G1 to S) for example:
DNA damage à p53 activation à p21 expression à inhibition of cyclin E/CDK2 à block of
transition to S phase.
APC complex – triggers the separation of the sister chromatids by promoting the destruction
of cohesins (proteins that hold two chromatids together) and activation of separase.
MPF complex – regulates contractile ring formation.
Topoisomerase is a protein that creates double-stranded breaks in DNA to manage DNA
tangles. It plays role in chromatin decondensation releasing the tension during the tension
during the untangling.

Replication – two identical double-helixes are synthesized from one original double strand
1. This process is initiated by a protein called DNA helicase which moves along the DNA
strand and binds to the special site called origin of replication. Once bound, it moves
along the DNA strand towards both sides creating the replication fork, unwinding the
strands and separating them.
2. Once separated, the strands naturally what to return to the original position of
binding back since the intermolecular hydrogen bonds attract the strands to each
other. In order to prevent this, enzymes called single strand binding proteins (SSBP)
bind to the bases, therefore, keep them from re-associating.
3. Then the enzyme called primase forms an RNA primer, which signals for the DNA
polymerase to begin process of taking up free nucleotides and assembling them to
the single strand while connecting via phosphodiester bonds. DNA polymerase can
only synthesize the strand in the 5’à3’direction due to the chemical composition of
the enzyme. Also, it cannot start synthesizing the strand without the RNA template.
4. DNA polymerase is kept attached to the strand by a protein called sliding clamp.
5. The synthesis of leading strand is carried out continuously since DNA polymerase
has no problem in synthesizing the strand in that direction, whereas the synthesis of
the lagging strand is carried out discontinuously, in pieces called Okazaki fragments.
Although the direction of that strand is opposite from what the DNA polymerase

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more comfortable with, each Okazaki fragment is still synthesized in the 5’à3’
direction, but in shorter pieces.
6. After, the individual pieces are joined together by enzyme ligase
Proofreading – correction the work of the DNA polymerase. This process, contrary to
replication can be carried out only in the 3’à5’direction. This work carried by DNA
polymerase – therefore it is both synthesizing new strands as well as proofreading itself. In
case of binding of a wrong nucleotide, DNA polymerize removes it and substitutes to the
correct one
Mismatch repair – correcting the mistakes of the replication machinery. Special proteins
identify the mismatched pairs due to the deformation of the helix, since the bases are
mistaken. They remove the wrong base, then DNA polymerase inserts the correct ones.

Transcription
The process of forming mRNA from the DNA (mRNA then takes the genetic information out
of the nucleus into the cytoplasm. Although RNA and DNA are pretty similar, in RNA uracil
binds adenine instead of the A=T bond. Transcription occurs in several important steps:
1. Initiation. A group of proteins called initiator factors move along the DNA until they
find the promotor region. Then the promotor region signals for the RNA polymerase
to begin synthesis of RNA while unwinding the DNA strand.
2. Elongation. RNA polymerase starts synthesis of the new RNA strand and assemble
nucleotides connecting them with the phosphodiester bonds
3. Termination. Eventually, RNA polymerase reaches the termination region and after
that, special proteins assist in the dissociation process.
Completed mRNA then undergoes series of posttranslational modifications:
- RNA capping (7-methylguanosine is bound to 5’of the RNA)
- RNA polyadenylation (100-200 repeated adenine nucleotides are bound to the 3’of
mRNA)
- RNA splicing – removal of introns by enzyme called spliceosome, which contains
snRNAs that recognize boundaries between introns and exons.
- Alternative splicing (optional) not only introns are removed, but also some exons – all
to produce various types of RNAs.
Regulation is carried out by activators and enchanters as well as general transcription
factors bound to the special region called TATA box in the gene.
There are some proteins which are regulating transcription which include homeodomain,
zinc finger, leucine zipper.

Translation – formation of the protein from mature mRNA. Then new polypeptide chain is
synthesized from the NàC terminus.
mRNA is used as a template by the ribosomes to synthesize proteins. Ans it is read by the
codons (triplets of nucleotides).

So, mRNA carries the genetic info from the nucleus to the cytoplasm, rRNA uses mRNA as a
template to synthesize proteins and tRNA takes amino acids from the cytoplasm and binds
them to the ribosomes for the protein synthesis. Translation is carried out in three steps:
1. Initiation. mRNA travels from the nucleus into the cytoplasm of the cell. Then small
unit of the ribosome binds to the 5’ end by the recognition of the cap and moves
along it till it reaches the start codon (AUG – methionine). Once tRNA with

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methionine binds to the start codon, large subunit comes and binds with the small
subunit à completes the ribosome.
2. Elongation. When elongation begins, the P site is at the start codon (AUG). Then the
next tRNA comes with the new amino acid and binds it to the A site. There is also a
special enzyme inside the ribosome called peptidyl transferase that creates the
peptide bond between the amino acids and only after the amino acid is released
from the P site. After those steps are complete, the ribosome moves towards the 3’
and the tRNA which was attached to the P site moves to the E (exit) site and gets
released. This process continues till the process is complete.
3. Termination. When the ribosome reads either one of the three stop codons (UGA,
UAA, UAG), a protein called release factor binds to the A site and causes the
polypeptide chain to break off at the P site. The ribosome then dissociates and the
translation ends.

Cell senescence and differentiation

Differentiation – the process of generating diverse types of cells (specialization of cells)
Determination – process in which the cell becomes committed to a certain fate. The cell is
usually determined before it starts to differentiate.
Since all the cells in the body contain same number and variety of genes, our bodies created
a nice way out how to have cells with different material play different role in the body – by
expressing different genes (it means that only needed genes are expressed, whereas not
needed are just chilling passively on the chromosomes.

Homeotic genes are genes regulating the development of anatomical structures in various
organisms. Hox genes are genes that control the body plan of the embryo along the cranio-
caudal axis. Those genes function as molecular markers if the body region. All of the hox
genes are located on different chromosomes and expressed independently. – Hox A,B,C,D.

After a particular cell realized a certain number of the cell divisions (hayflick limit), it looses
its ability to proliferate and dies, this process is called cell senescence.
Hayflick limit determines the number of cell cycles that the cell can undergo. It applies for
almost all cells in the body. Though stem cells, germ cells and cancer cells do not have this
limit.

Cell senescence is influenced by many factors, such as

- Oxidative damage (reactive oxygen species)
- Accumulation of mutations
- Damage of mitochondrial genome
- Damage of insulin signaling, since insulin regulates basal metabolism
- Telomere shortening – that way p53 is activated as the cell recognizes telomere
shortening as DNA damage.
Cell death – can be divided into 2 distinct groups –
o Apoptosis – programmed cell death which requires energy, occurs without
inflammation, the plasma membrane stays intact while the cell itself is
shrinking and chromatin decondenses and DNA is being degraded.

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o Necrosis – cell death due to irreversible cell damage, occurs with

inflammation, cell membrane swells and disintegrates while Chromatin and
DNA stays intact.

Regulation – can be realized via both endogenic and exogenic signals.

o Endogenous:
Tumor-suppressor gene p53. The key molecule in the cell cycle arrest and apoptosis or
senescence. It induces activity of mdm2 (feedback inhibition of p53), p21 gene (blocks cell
cycle by inhibiting CDK2), Bax gene (induces apoptosis), IGF-BP3 (blocks IGF-1 activity)
o Exogenous:
Apoptotic signal molecules
Glucocorticoids (cortisol) – induces Bcl2 (Bax)
Cytokines (viability factors) – inhibit proapoptotic proteins (IGF-1, IL-3, IL-4, IL-6, GM-CSF
Ligands of death domain receptors – TNF, Fas ligand, TRAIL ligand
Granzyme B – enzyme found in cytotoxic T cells and NK cells. It secretes perforin which
perforates the cell membrane causing its leakage plus allowing granzyme to enter the cell
and activate caspase 3 to kill the cell.

Bcl family is a family of proteins that regulate apoptosis by either initiating or inhibiting it.
Proapoptotic proteins: Bax, Bak, Bad, Bid, Puma
Antiapoptotic: Bcl-2, Bcl-XL

Pathways:
- Mitochondrial aka intricsic pathway
P53 activation à Bax expression à Bax channels opening in the mito membrane à
cytochrome C release from mitochondria à apoptosome formation à caspase 9 (initiator
caspase) activation à caspase 3 (executioner caspase) activation à APOPTOSIS

- Death domain aka extrinsic pathway

Ligand binds to receptor with the death domain (Fas) à formation of the DISC complex à
caspase 8 (initiator caspase) activation à caspase 3 (executioner caspase) activation à
cleavage of the death substrate à apoptosis

Caspases are cysteine proteases that initiate apoptosis. Activated by proteolytic cleavage.
- Initiator caspases (caspase 9, caspase 8, caspase 2)
- Executioner caspases (caspase 3, caspase 6, caspase 7)
- Inflammatory response caspases (caspase 1)
When executioner caspases cleave death substrates, apoptosis occurs. Death substrates
include: PARP – only cleaved PARP initiates apoptosis, CAD, cytoskeletal proteins, Rb protein.

Renewal and repair

In the body we can find two types of cells – those that can regenerate and permanent cells
(those that cannot proliferate and in case of injury cannot be fixed). Though those that can
regenerate can be renewed or can be repaired.
Cells can be renewed from either already differentiated cells or undifferentiated progenitor
cells (those that come from stem cells, but still more differentiated than the stem cell
(undifferentiated cells that are capable of differentiating throughout the whole life of the

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organism, they also maintain the pool of stem cells, while dividing into 2 cells one goes into
the further development and the other stays undifferentiated)). Those processes are
regulated by cytokines.

Cells can be repaired from repair regeneration process (when the original function and
morphology is kept) (this process is very limited and available in only small amounts of
internal tissues) or wound healing (replacing of the damaged cells by the connective tissue –
loosing the function and morphology of the original tissues)

Compensatory hyperplasia is when a damage of the internal organ is fixed by proliferating

of the undamaged part of it – in the liver
Compensatory hypertrophy – in case of damage of a paired organ, the other organ is
proliferating more, therefore substituting work for both – in kidneys

Viruses – small infectious agent that replicates only inside of the living cells of another
organism. Viruses can affect all of the life forms. There are two terms that specialists use
special for viruses: Virulence (ability of virus to cause the disease – for example,
bacteriophages have no virulence in humans, but very high virulence in bacteria) and
invasicity (ability of the virus to penetrate through the tissue and destroy it)
When virus is not inside of the cell yet, it exists as independent particle, aka virions (naked
viruses)
Structure of virus:
1. Nucleic acid – DNA or RNA
2. Protein coat surrounding the genetic material, aka capsid (composed of proteins
capsomeres)
3. Envelope – surrounds the capsid when the virus is outside of the cell. Most of the
time envelope is formed from the cellular membrane, therefore, contains cellular
membrane and cellular phospholipids.
Viruses have no ribosomes.
There are also some subviral particles which can also promote infections.
- Viroids – free chains of RNA that are able to replicate inside the cell and cause
diseases
- Virusoids – the parasites of viruses that can code proteins. Those are not entirely
autonomous agents as they can only replicate in the presence of a virus inside the
cell (so, basically they cannot cause infection on their own but can make already
existing infection much worse)

Since viruses cannot live and grow on their own, they have several ways to make it inside the
cell:
- The virion is getting absorbed on the surface of the cell (membraned of the cell and
viral envelope fuse, then the nucleocapsid of the virus is released into the cell’s
cytoplasm) (in bacteriophages: DNA is injected directly into the cytoplasm of the
bacteria)
- Viral macromolecules are being produced (nucleic acid is released from capsid and
then replicated)
- Mature virions are released from the cells by either cell lysis – necrosis, or by
budding mechanism – exocytosis of virions

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Viruses, depending on the type can replicate in the nucleus or in the cytoplasm of the host
cell
Also, depending on the type of virus the consequences of viral invasion can be either death
by necrosis/apoptosis, reversible damage or no injury at all

Viral types are divided by their replication strategy:

- Ds DNA virus
o Papillomaviruses (HPV)
o Herpesviruses (herpes and also in case of Epstein Barr virus – Burkitts
lymphoma)
o Poxyviruses (small pox, chicken pox)
o Hepadnavirus (hepatitis B)
- +ss RNA virus
o Picornaviruses (polio, hepatitis A, rhino viruses)
o Hepatitis C virus
o Rubeola
o Retroviruses (HIV, Human T-cell leukemia virus 1, Kaposhi sarcoma)
- -ss RNA virus
o Orthomychoviruses (influenza)
o Paramyxoviruses (measles and mumps)
o Rabdoviruses (rabies)
o Filoviruses (ebola)
- Ds DNA virus
o Reoviruses
Viruses cannot only damage the cell immediately and completely, they can also be latent –
the genome is inside of the host cell, but the viral proteins are not expressed, or viruses can
be persistent – the viral genome is inside of the cell, but only small part of proteins are
produced

Cancer genes – genes coding for proteins with their function changed to development of
tumors (cell transformation)

Oncogenes (AD)
Genes that are by means of their products (oncoproteins) transform cell. They are derived
from protooncogenes by change of the structure of protooncogene or the increase of the
protooncogene expression. This can happen by means of point mutations, deletions, gene
amplifications, translocations, formation of fused genes. Also they can originate from the
invasion of the virus (if they are from the RNA viruses, then oncogenes originate from cell’s
oncogenes, but if from DNA viruses, then the nature of the oncogene is viral)
Tumor suppressor genes (aka antioncogenes) (AR)
Those genes are by means of their products block cell transformation. The loss of function
of tumor suppressor genes can be because of the deletions, chromosome loss, epigenetic
changes or mitotic recombination (which mistakenly transport this part of the gene into the
intron region and it becomes silent). The most important tumor suppressor genes are p53,
Rb gene and CDK inhibitors, such as p21 and p16 genes.
Genes of the DNA repair (AR)

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Those genes code for proteins which are involved in the DNA repair. Loss of their function
results in accumulation of genetic changes.

Cell transformation and tumor cells

Cell transformation of the process of formation of a tumor cell out of normal cell.
Tumor cells are immortal, less differentiated then normal cells, able to spread and form
metastasis in other tissues and organs, activating oncogenes in the nearby cells, have
increased proliferation, lack of contact inhibition (therefore are very closely packed
together) and lack anchorage dependence (can move easily), have decreased apoptotic
activity, have clonal growth, have their own blood supply, use glycolytic pathways for their
energy supply, have impaired DNA repair.
Tumors in general can be Benign (those that grow rapidly, although are non-invasive and
stay in place) or Malignant (those that are capable of growing into the other tissues)

Cells can become transformed by means of several mechanisms which include point
mutations, deletions, gene amplifications, translocations, formation of fused genes.
Also cells can be transformed by external agents that are causing cell transformations. They
can be:
- Chemical (that cause point mutations)
- Physical (radiation) – UV radiation causes thymine dimers and ionizing radiation
causes chromosomal translocations
- Viruses
Those genetic changes can lead to activation of oncogenes, inactivation of tumor
suppressor genes or inactivation of genes responsible for the DNA repair

Genetics
Mutagens and carcinogens of the environment
The mutagens and carcinogens can be divided into 2 distinct parts:
1. Environmental, aka exogenous, factors which includes
a. Ionizing radiation (affects both chromatids, may form dicentric
chromosomes, ring chromosomes, defragmentation)
b. UV radiation (creates thymine dimers which affect transcription and
translation)
c. Chemicals (affect only one chromatid causing chromatid breaks and
exchanges)
2. Endogenous damage which includes
a. Hydrolysis which cleaves base from the DNA strand
b. Deamination
c. Methylation
d. Oxidation
e. DNA breaks
f. Replication breaks which were not detected by proofreading

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Though our genetic material is so prone to damages, there are several ways that our cells fix
those problems by repairing DNA
1. direct reversal, aka direct ligation, demethylation.
2. Single strand breaks repair:
a. Base excision repair (BER): enzyme glycosylase recognizes the damaged
base, then enzyme endonuclease comes and removes the damaged site, then
the gap is filled by DNA polymerase-betta and sealed by ligase
b. Nucleotide excision pair (NER): enzyme endonuclease recognizes damaged
nucleotide, then the oligonucleotide at the damaged site is removed by
exonuclease, and finally, DNA polymerase fills the gap and the site is sealed
by ligase
c. Mismatch repair (MR): endonuclease recognizes the mismatched part,
exonuclease then removes the wrongly-placed nucleotides, DNA
polymerase-delta fills the gap and ligase seals everything
3. Double-stranded breaks repair:
a. Non-homologous ends joining (NHEJ) – connection of the ends which are
non-homologous
b. Homologous recombination (HR) – this type of repair is realized only in case
of presence of a sister chromatid or a homologous chromosome. A special
protein searches for the homology and joining homologous ends, then DNA
polymerase fills the gaps
c. Single strand annealing (SSA) – endonuclease digests both strands of broken
ends to leave single stranded tails, the tails then search for the homologs
among each other and then everything is sealed by ligase.

Chromosomes and tumors

Human chromosomes have certain sites which are unstable and more prone to mutations
comparing to the other sites. Those are called fragile sites. There is a list of diseases that are
important for the exam:

Chronic myelogous leukemia

Fusion of chromosomes 22 (Abl gene) and 9 (Bcr gene). The fusion of those two genes cause
formation of the Philadelphia chromosome. SYNTHESIS OF THE ABNORMAL PRODUCT. This
is treated by the tyrosine kinases inhibitors

Burkitt lymphoma
Translocation of c-myc gene between 8 and 14 chromosomes. OVERPRODUCTION OF
NORMAL PRODUCT

Retinoblastoma (AD with reduced penetrance)

Inactivation of the tumor suppressor gene Rb. For the manifestation of the disease
inactivation of both of the alleles is required. Retinoblastoma can be divided into 2 types:
- Heritable – one mutant allele is inherited from a parent and the other one occurs by
a somatic mutation. The onset is usually early in life and both of the eyes are affected
- Sporadic – somatic inactivation of both of the alleles. This type manifests itself later
in life.

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Wilms tumor – kidney cancer

Mutation or deletion of the WT1 gene in the region of the 11p13 gene

Chromosome instability syndromes (AR inheritance)

Fanconi anemia – caused by breaks and chromatid exchanges as well as defect in DNA
repair. Manifested as too few blood cells due to the bone marrow function failure, growth
retardation, small head and hyperpigmentation

Bloom syndrome – caused by breaks and exchanges between homologs and sister
chromatids which result in defect of replication and reparation. Associated with low birth
weight, very slow growth, immunodeficiency and extreme sun sensitivity

Ataxia telangiectasia – caused by rearrangement of chromosomes 7/14 or 2/22 which

causes defects in the DNA repair. Associated with growth retardation, dysfunctional muscles,
immunodeficiency and café-au-lait spots on the skin

Xeroderma pigmentosum – caused by defects in DNA repair and manifested as extreme

sensitivity to radiation which results in various skin cancers

Nijmegen breakage syndrome – caused by rearrangement of chromosomes 7/14 which

results in defect of double – stranded DNA breaks repair. Manifested as growth retardation,
mental retardation, immunodeficiency and small head

Werner syndrome (premature aging) – caused by defects of endonuclease and helicase

activity. Manifests as formation of cataracts, calcification of the skin, premature grey hair
and premature atherosclerosis

Cockayne syndrome (premature aging) – caused by defects in DNA excision repair.

Manifests as slow growth, mental retardation, deafness.

Li Fraumeni syndrome – mutation of p53 (tumor suppressor gene)

Epigenetics
Epigenetics is the change of gene expression without changing the nucleotide sequence! It
can be heritable!
As any other modification, the change of epigenetics depends on dose, age, exposure, sex
etc.
Types:
- DNA methylation of CpG islands (methylation «hot spots»)
- Histone modifications
- Inactivation:
- Deacetylation (H3K9) (histone deacetylase)
- Demethylation (H3K9, H3K27, H4K20) (histone demethylase)
- Sumoylation
- Ubiquitination

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- Activation:
- Acetylation (H3K9) (histone acetyltransferase)
- Methylation (H3K4, H3K36, H3K79) (histone methyltransferase)
- Phosphorylation
- Ubiquitination
- RNA interference - gene expression silencing
- Short RNAs are inactivated from viruses or transposons
- Types:
- Micro RNA (miRNA) - binds to target mRNA and inhibits
translation
- Short interfering RNA (siRNA) - initiation of mRNA
digestion/degradation
- Piwi-interacting RNA (piRNA) - influence of epigenetic
regulation, control of transposon silencing
- Long RNAs inactivated the X chromosome
- Chromosomal position effect
- insulators: sequences along DNA that prevent distal enhancers from
activating/silencing genes
- Polycomb and Trithorax complexes - maintain expression states (active or
silent) of homeotic genes.
- Prions and prion-like phenomena
Metastable epialleles are identical alleles that due to epigenetic modifications are variably
expressed

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Biochemistry
Metabolism of nucleic acids

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Biotransformation of xenobiotics
Xenobiotics are the foreign substances found within the body that is not naturally produced
or expected to be found within the body.
Xenobiotics are very often lipophilic substances and can enter cell by passive diffusion or
transport systems such as pinocytosis or phagocytosis
In order to get rid of them, they have to be transformed – converted into more polar
substance and it happens in two phases:
1. Biotransformation – hydroxylation or reduction hydrolysis – to increase the polarity
2. Conjugation – synthesis of small substances with polar functional groups – to
increase polarity even more, therefore not allowing them to penetrate biological
membranes, and lets the substances out with the urine or bile.
Glucuronic acid is the most known conjugation agent that transforms xenobiotics within the
conjugation phase.
Cytochrome p450 (CYP) are a family of proteins containing heme as a cofactor, those
enzymes are responsible for 90% of drug excretion from the body

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Biochemistry of aging
Oxidative stress – imbalance between formation and elimination of ROS and RNS that
causes the excessive production of radicals or improper antioxidation.
ROS include:
- Superoxide à H2O2 + molecular oxygen
- Ascorbic acid à semi-hydro-ascorbic acid
- O2 + e- à superoxide
- O2 + 2e- à peroxide
Fenton reaction
Fe2+ + H2O2 = Fe3+ + hydroxyl radical + hydroxyl ion
Ferritin – protein that keeps iron in the oxidized state
Albumin – protein that transfers Cu in blood plasma
Ceruloplasmin – protein that transfers Cu in blood plasma as well as binds free hemoglobin

Low molecular weight antioxidants

WATER SOLUBLE
- Ascorbic acid (vit C – exogenous)
- Glutathione (endogenous)
- Uric acid (endogenous)
- Lipoic acid (endogenous)
FAT SOLUBLE
- Carotenoids + vitamin A
- Alpha-torophenol (vit E)
- Ubiquinol (CoQ)

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Embryology and histology

Fertilization
A sexually mature female produces an oocyte which is released from the ovary by the
rupture of the Graafian follicle. After the follicle is released into the oviduct, the remain
structures of the Graafian follicle develop into the temporary endocrinological organ –
Corpus luteum, which produces progesterone.
By the time the oocyte is in the ovarian tube, it still did not complete it’s meiotic division and
it is arrested in the meiotic metaphase II.

A sexually mature male ejaculates into the vagina and his sperms travel through the cervix
of the uterus and uterine cavity into the fallopian tube as well to meet the oocyte. The fastest
sperm reaches the oocyte and binds a structure called zona pellucida. Then in the sperm
the acrosomal reaction takes place and the sperm penetrates zona pellucida and the sperm
membrane fuses with the oocyte membrane, while all of the genetic material together with
all the contents goes inside the oocyte.
In order to prevent polyspermy, oocyte release special cortical granules.
So, after penetration by the sperm, the genetic material of the oocyte is finishing the
meiotic division forming a female pronucleus and a second polar body.
Then, the female and male pronuclei fuse together into a METAPHASE OF FIRST MITOTIC
DIVISION initiating the cleavage. Now this structure is called zygote.
Important to notice, healthy fertilization takes place in the ampulla (the widest place) of the
oviduct.

Cleavage and blastocyst formation

As the zygote moves slowly through the fallopian tube, its cells divides equally and very fast,
though the zygote is not changing its shape and size due to zona pellucida which prevents
enlarging of the structure.
After three days of moving through the fallopian tube the zygote is already a ball of cells,
which are equal and the structure is called morula. Morula is released from the fallopian
tube into the uterine cavity and just flows around for a couple of days.
Since the inside of the uterus is very moist, since uterine glands produce a lot of glycogen,
lipids and water, also the number of cells in the morula grows progressively and they need
more and more nutrition to survive, the cells in the morula start to rearrange themselves
for a more productive nutritional purpose. So, the cells, which were more towards the poles
of the morula become the outer cell mass - those cells are not very close to each other and
allow the liquid to flow through by diffusion, and the inner cell mass, those cells become
very closely packed and have a lot of gap junctions in between them, allowing the nutrients
to get inside every single cells. Therefore, since the cells are now not as equal, the structure
is called early blastocyst. The inner cell mass is packing itself closer together, simultaneously
moving to one pole, creating a cavity in between the inner cell mass and the outer cell mass.
This cavity is called blastocyst cavity.

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Even though the cells are more or cell specialized, and the structure is called early
blastocyst, it is still surrounded by zona pellucida.
After approximately 5 days after fertilization the blastocyst is big enough and it starts to
hatch out hatch out the zona pellucida. After hatching, the blastocyst is called late
blastocyst. The late blastocyst is already able to implant itself into the uterine cavity.

Implantation
Blastocyst is flowing around the uterine cavity while expressing special adhesive proteins
called selectins on the surface, those proteins are extremely attracted to the sugars which
are expressed on the surface of endometrium. When blastocyst attaches to the uterine wall
via those selectins, it starts to express another type of proteins on its surface which are
providing much stronger connection with the endometrium – integrins. Blastocyst normally
implants into the anterior or posterior wall of pars functionalis of endometrium.
When blastocyst attaches to the endometrium, it the outer cell mass – aka trophoblast –
start to secrete special proteolytic enzymes which are basically digging the hole inside the
endometrium finding its way to implant inside to get more nutrients and also to be more
protected. While the proteolytic enzymes are expressed, the endometrium undergoes a very
important change called decidua reaction, the pregnant endometrium, aka decidua, and its
vessels start to swell and enlarge to bring more nutrients to the baby and also decidual
glands enlarge and start to produce more and more glycogen and lipids for the baby
nutrition.
By this time, first week of development is complete and the second week starts. The second
week of development has a rule of 2 (basically everything what originates spits in 2)
So, trophoblast starts to differentiate into 2 structures:
- cytotrophoblast – cuboidal cells which are rapidly differentiating
- syncytiotrophoblst – actually those proliferating cytotrophoblastic cells move to the
periphery, but since the process is so rapid, they loose their boundaries and are seen
as just multinucleated mush which expresses proteolytic enzymes for further
implantation as well as expresses hCG (which actually causes morning sickness in the
first trimester.

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The inner cell mass – aka embryoblast, also splits into two structures
- columnar upper cell layer – epiblast
- squamous cell layer beneath it – hypoblast.
Further both of those layers proliferate forming amniotic cavity from epiblast above and
primitive yolk sac from hypoblast below.

Trophoblast differentiation:
Synsytiotrophoblast keeps digging into the pars functionalis of the endometrium and at
some point it starts to create little empty spaces – lacunae inside the cell mass. Also it grows
around uterine vessels and glands surrounding them. Then, syncytium expresses the same
proteolytic enzymes onto the vessel walls, therefore perforating it. And mothers blood leak
into those lacunae, therefore, providing good nutrition to the cells.

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Meanwhile, syncytiotrophoblast together with cytotrophoblast proliferate in the papillary –

projection pattern forming villi. Villi which consist only of cytotrophoblast and syncytium are
called primary villi.

How the blood exchange works: in the human body arteries carry out blood having a very
high pressure in them, but veins, in the opposite, have very low pressure in them. Since
syncytium perforates both types of the vessels, blood from the arteries are pumped inside
the lacunae with the very high pressure, while veins are basically sucking up the blood from
the lacunae because of the low pressure in them. Therefore, the blood in the lacunae is
renewed constantly.

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Embryoblast differentiation:
Cells of the hypoblast, which are forming the primitive yolk sac proliferate even more but
they change their appearance into the mesenchyme-appearance cells. Those cells move all
around the primitive yolk sac and amniotic cavity just beneath the cytotrophoblast forming
the mesenchymal layer and occupying all the space. Those cells are called extraembryonic
mesoderm. So, the cells proliferate more and more and eventually become a pretty thick
layer of mesenchyme. Since they need nutrients as well, which at that time comes by
diffusion in the form of a solution, they start firming cavities which eventually fuse between
each other forming two layers of mesoderm (splanchnic extraembryonic mesoderm adjacent
to the yolk sac and the amniotic cavity and somatic extraembryonic mesoderm adjacent to
cytotrophoblast) and the cavity in between those two layers called chorionic cavity. somatic
extraembryonic mesoderm together with cytotrophoblast and syncytium are forming a
structure called chorionic plate.

The cavity forms everywhere, except one particular part, near the amniotic cavity, where
those two layers stay connected to each other. This connection is called connecting stalk
and it will further develop into the umbilical cord.

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Hypoblast adjacent to epiblast are forming the bilaminar disc. They are located very closely
together, therefore, no mesoderm is formed in between them.

When the third week of the development starts, the structure called primitive streak starts
to form on the surface of epiblast. Initially, it is very hardly defined, but as it grows and
develops, it starts to be seen as the groove with the elevations in both if the sides from the
groove. Primitive streak is the first and initial structure that determines the axis of the
embryo.

The end of the streak that moves forward is called primitive node, defining the cranial end
of the embryo. The primitive node consists of a small invagination in the middle called
primitive pit surrounded by small elevated areas. Also, another structure appears in the
cephalic region of the embryo – oropharyngeal membrane – future site of the mouth. Cells
at that area are connected extremely tight between each other, no mesoderm is formed
between them!
Cells of the epiblast move inwards, “diving” inside the primitive streak, and change their
shape into the flask-shaped form, forming intraembryonic mesoderm. They detach from the
epiblast and slip beneath it. This process is controlled by FGF-8 which is synthesized by the
streak.

Once sufficient number of cells invaginated, some of them replace hypoblast forming
endoderm, the middle layer of cells forms mesoderm and the remaining superficial layer
forms exoderm. The cells which move through the primitive node in the cranial direction
form a new structure called prechordal plate.

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The cells of the prechordal plate move cranially till they reach oropharyngeal membrane
and as the association of endoderm and exoderm in that area is very tight, they get mushed
between each other forming notochordal plate. Once they reach oropharyngeal
membrane, they detach from ectoderm and form definitive notochord.

Notochord functions as the “spine” of the embryo and transfers signals during the
development, later it differentiates into muscles and some parts of the vertebra. At the point
of primitive pit, the pit itself perforates forming a structure called neuroenteric canal which
connects the amniotic cavity and the yolk sac together for a little bit of time. If in the cranial
end of the embryo there is an oropharyngeal membrane, on the caudal end there is a similar
structure called cloacal membrane which will in time give rise to the anus. Just near the
cloacal membrane the yolk sac invaginates into the connecting stalk and forms allantois.

The development of the CNS is controlled by Shh from notochord.

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The parts of the ectoderm thicken, forming a new structure called neural plate. It starts from
the middle portion of the embryo and extends into the cranio-caudal directions. In the 3rd
week of development the neural plate starts to fold, forming neural tube.

Neural tube also starts to fold from the middle portion and continues towards the cranio-
caudal directions. The neural tube is the precursor of the CNS. During the fusion of the tube
some cells on the crest of the neural fold detach, forming a new cell population – neural
crest. They give rise to the PNS.

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The anterior end of the tube is called anterior neuropore and it closes at ED25, whereas the
posterior end of the tube is called posterior neuropore and it closes at ED27. Lack if folic acid
during pregnancy causes non-closure of either neuropores and can lead to anencephaly or
spina bifida.

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After the successful closure of the neural tube on the anterior part three swellings appear.
They give rise to the future brain.
- Proencephalon (forebrain)
o telenceplalon
o diencephalon
- Mesencephalon (middle brain)
- Rhoencephalon (hindbrain)
o midencephalon
o myencephalon
The rest of the neural tube forms spinal cord. And the signaling comes from the notochord.

On the sides of the neural tubes the mesoderm – paraxial mesoderm creates accumulations
– bubbles which are called somites. First pair of somites appear at the end of the third week
in the occipital area and then appear in pairs extending in the caudo-cranial directions. In
total there are around 42-44 pairs of somites. In the cranial region somites are called
somitomeres. They appear prominent during 4th and 5th week of the development,
therefore, the doctors count those structures in order to determine the age of the embryo.

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Placenta.
Placenta consists of fetal part (chorionic plate and villi) and maternal part (endometrium -
pars basalis and pars functionalis)
Development of the placenta starts at the point of implantation. Syncitiotrophoblast
secretes hCG in order for corpus luteum not to degenerate and secrete progesterone.

Syncitiotrophoblast secretes proteolytic enzymes and creates finger-like projections in the

endometrium.
Lacunae within syncytium are getting filled with the maternal blood and glandular
secretions, they fuse together forming the network of lacunae and establishing early
uteroplacental circulation.
Towards the end of the 2nd week small projections of cytotrophoblast invade syncytium
forming primary chorionic villi. Early in the 3rd week extraembryonic mesoderm grows into
the primary villi forming the core of loose connective tissue – secondary chorionic villi. By
the end of the 3rd week blood islets (which originated in the yolk sac) migrate throughout
mesoderm forming capillaries and start to appear in the secondary chorionic villi. Chorionic
villi with its own circulation are already tertiary chorionic villi.

Cytotrophoblast of some of the tertiary villi grows forward beneath decidua basalis and
spread across, forming a shell.

By the 4th week fetal blood flow is fully established and two umbilical arteries and one vein
are formed. Maternal blood, better say, nutrients from maternal blood, diffuses from the
lacunar intervillous spaces through the layer of syncytium, cytotrophoblast, extraembryonic
mesoderm and endothelium of capilaries and gets into fetal blood flow.

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The spaces between the anchoring villi are called cotyledons and there are 15-20 of them in
the end of pregnancy.

Placental membrane: is the membrane that separates maternal circulation from the fetal
circulation. It consists of syncytium, cytotrophoblast, connective tissue and epithelium of villi.
By the end of pregnancy though cytotrophoblast is regressed – just to create a more
efficient diffusion between mother and the child.
When placenta is fully developed, it takes the role of production of progesterone instead of
corpus luteum. Also, maternal antibodies can cross the placenta to create some kind of
immunity for the baby.

Pharyngeal arches
1st Mandibular arch – gives rise to the muscles of the lower jaw
2nd Gives rise to all of the muscles of facial expressions
3rd Gives rise to muscle that elevates larynx and pharynx
4th Gives rise to all the muscles of the palate as well as muscles of the pharynx
5th and 6th give rise to some muscles of the palate and pharynx
They all are derived from ectoderm and start to develop during 4th week. Each pharyngeal
arch consists of endoderm, mesoderm, and endoderm and also the cranial nerve, an
artery, muscle tissue and cartilage. The arches are separated by pharyngeal clefts.

Derivatives of the germ layers:

Endoderm:
Digestive tube and respiratory system, thyroid gland, parathyroid gland and thymus,
pancreas and liver, epithelium of urinary bladder, urethra and vagina

Mesoderm
Gives rise to the connective tissue (cartilage, bone, muscles, mesothelium), kidneys, genital
system, cortex of adrenal gland and vessels.

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Ectoderm
Gives rise to the nervous system (CNS, PNS, and nerve cells), retina of the eye, epidermis,
nails, hair, mammary glands, medulla of adrenal gland, cells of the neural crest ( ganglia,
Schwann cells, melanocytes, medulla of suprarenal gland) and enamel.

Somites
They originate from paraxial mesoderm. When they differentiate, they are affected by some
signaling molecules. So they differentiate into: dermatome, myotome (muscle tissue) and
sclerotome (vertebra)

Development by week:
4th week:
- closure of neuropores (ED24 & ED26)
- formation of somites (4th-12th days)
- pharyngeal arches (visible after ED26)
- heart can be seen
- developing of upper limb buds (ED27-27)
- otic (ear) pits and lens placodes
- lower limb buds – by the end of the week

5th week:
- head growth – brain and pharyngeal arches
- development of face and neck
- ectodermal grooves, endodermal pouches, pharyngeal membranes
- aortic arches – vessels in pharyngeal arches

6th week:
- development of limbs – future fingers
- development of the ears
- head is larger than the whole body

7th week:
- fingers appear on the limbs
- umbilical herniation – intestine enters the extraembryonic coelom in the proximal
region of the umbilical cord

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8th week:
- development of toes
- tail fully disappears
- body grows – head is now approximately half of the embryo
- eyelids fuse by the end of the week
- development of external genitalia
- apoptosis of tissues in between the fingers and final formation
- ossification in femur

Signaling during development:

Types:
- induction à tissues react according to the signals from other adjacent tissues
- reciprocal induction à tissues exchange information

1. body axis signaling

a. left-right – depends on the concentration of the morphogens
b. ventro-dorsal – FGF-8 produced by the primitive node and primitive streak
2. limbs
a. same principle as the somite development
b. caudal signaling center produces Shh and retinoic acid
c. Apical ectodermal ridge (AER) produces FGF and BMP4
d. Different concentration of morphogens causes specific expression of Hox
genes which causes specific differentiation
e. BMP induces skeleton formation
i. Axis of limb development
1. AER (apical ectodermal ridge) – proximo-distal axis (FGF
induces proliferation)
2. ZPA (zone of polarizing activity) – anterior-posterior axis – Shh
3. Ventro-dorsal axis – Wnt-7
3. Somites
a. Wnt and BMP4 – myoblast differentiation
b. Shh and noggin – sclerotome
c. Development of somites is regulated by notochord, neural tube, mesoderm
and ectoderm.
d. The gradient of morphogens from the notochord to the surface ectoderm
causes differentiation of somites to the scletotome and dermamyotome
4. Eye
a. Reciprocal induction between the superficial ectoderm and the eye cup –
development of lens and retina
5. Kidneys
a. Wnt and BMP7 causes branching of ureter bud
b. FGF – proliferation of mesenchymal cells
c. BMP7 and GNDF induces the transformation of the mesenchyme to the
epithelial cells
6. Vessels

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a. VEGF – stimulates the blood islet formation of the primary capillary network
b. Angiopoetin – stimulates branching of the vessels
c. PDGF and TGF-beta stimulate wall differentiation

We have the cranial and caudal signaling centers:

Cranial consists of the definitive ventral endoderm and mesoderm which are adjacent to the
prechordal plate

Caudal consists of primitive node and primitive streak

- Produces Wnt, FGF, FGF8, Shh, retinoic acid

Signaling molecules:
1. Shh – sonic hedgehog – produced by cells of notochord.
a. Regulates growth of digits and limbs, organization of the brain, CNS
development, neuron migration, neural tube, somite and limb development.
b. Morphogen
c. Activated by cholesterol
2. Wnt – produced by primitive node
a. Regulated intracellular interactions during embryogenesis, cell fate
specialization, cell proliferation and migration, planar cell polarity,
development of neural tube and limb buds
b. Uses beta-catenin as a second messanger
c. Morphogen
d. Both paracrine and autocrine signaling
3. Notch – produced by primitive node
a. Involved in the renewal of the gut lining, maintenance of the stem cells
b. Lateral inhibition
4. Signaling molecules binding to the receptor of tyrosine kinases
a. FGF- regulation of embryonic development, wound healing, endocrine
signaling pathways

The simultaneous stimulation of retinoic acid and BMP4 contributes to the differentiation
of cell segmentation of the embryo

Hox genes are the molecular markers that control the body plan of the embryo along the
tead-tail axis.

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Developmental toxicology
drugs:
- thaliomide (treatment of nausea during pregnancy in the 60s) (critical period day 38-
50)
o cardiac and other malformations
- cytostatics (used in chemotherapy - inhibits proliferation)
- Warfarin and other coumadine derivatives (blood clotting)
o small nose, low IQ, caridical malformations, inner bleedings (CNS), ossification
impairment
- anti-epileptic drugs
o growth retardation, lower IQ, facial morphogenetic malformations
- retinoids and vitamin A
o impairment of CNS, low IQ, cardiac malformations, morphogenic
malformations
- alcohol
o growth retardation, face dyrmorphology, impairment of CNS, cardiac murmur
(=bilyd)
- androgens
o diethistilbestrol (hormonal drug for reproductive system)
o impairment of vaginal development, endocrine disruptor
- antagonists of folic acid
o growth retardation, calvary bone hyperplasia, face dysmorphology,
hypodactyly, syndactyly
- lithium
o used to treat bipolar disease
- ribavirin
- mycophenolate mophetil (antibiotic used after organ transplantation)
o impairment of ear development
- ACE inhibitors (hypertension treatment)
o impairment of kidney function —> kidney failure
- non-steroidal anti-inflammatory drugs
o effect on ductus arteiosus closure - increased bleeding

Chemical carcinogens:
• Organic:
- Polycyclic aromaic hydrocarbons - crated during incomplete combustion of carbon-
containing fuels
- benzo(a)pyrene
- naphtalene
- Aromatic amines
- Chlorinated biphenyls
- Azo compounds
- Epoxides
- Alfatoxins - very potent hepatocarcinogen - found in fungal contamination of
peanuts
- Nitrosamides - found in sigarette smoke

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• Inorganic:
- Arsenic
- Chromium (VI)
- Cadmium
- Nickel
• Metal and polymeric implantates
- asbestos

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