TOPIC: ERECTILE DYSFUNCTION
These can also lead to cardiovascular
What is erectile dysfunction? Differentiate erection from ejaculation and
ED from impotence
Erectile dysfunction is a medical condition wherein a man is unable to
achieve or sustain an erection enough to have sexual intercourse.
Erectile Dysfunction Premature Ejaculation
Problem in achieving or sustaining A man ejaculate too early which is
an erection in the usually a problem in the
PARASYMPATHETIC nervous SYMPATHETIC nervous system
system (Fight para makabuntis o Flight d
makabuntis)
A man who prematurely ejaculates may be able to sustain an erection. A
man who has erectile dysfunction might not be able to ejaculate, due to the
inability to get an erection, or maybe hold an erection long enough to eject
sperm.
Additional notes: Physiologically, erection is triggered by the
parasympathetic division of your ANS, causing NO (a vasodilator)
levels rise in the trabecular arteries and smooth muscles of the penis.
For this reason, arteries will dilate, causing the corpora cavernosa to
fill with blood. At the same time, ischiocavernosus and
bulbospongiosus muscles compress the veins of the corpora
cavernosa, restricting the egress and circulation of this blood.
Ejaculation is controlled by your sympathetic division.
What is the etiology and risk factors in ED?
Lifestyle - This is the etiology wherein the parameters can be
controlled. Examples are diet and exercise. Diet and exercise affects
BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E
blood flow and composition.
and endocrine diseases than can seriously disrupt the blood flow to
the penis. Type-II diabetes can lead to neurogenic erectile
dysfunction, and atherosclerosis from poor diet and no exercise can
disrupt blood flow with plaque, and this leads to vasculogenic ED.
Vices like smoking and drinking have adverse effects to the
cardiovascular and nervous systems as well.
Vasculogenic - Vasculogenic ED occurs when blood supply going to
the penis is disrupted. This can happen due to plaque buildup,
hardened arteries, and hypertension. In fact, erectile dysfunction
can be one of the symptoms of cardiovascular disease.
Neurogenic - Neurogenic erectile dysfunction occurs when nerve
supply leading to the penis is compromised. Parasympathetic nerves
are stimulated, and results to an erection. Lesions in the spinal cord,
nerve damage, and even stroke (which is considered to be vascular)
can affect the innervation of the penis. Another cause can be loss of
coordination in body movement (such as Parkinsons disease).
Psychogenic - Psychological disorders can also affect a mans ability
to achieve and sustain an erection. Depression and anxiety can alter
physiological and biochemical processes. For example, cortisol
increases in stress-filled situations, and prolonged cortisol exposure
can lead to increased cholesterol and truncal obesity that can cause
erectile dysfunction.
The risk factors in ED stem mainly from the lifestyle etiology of the disease.
Risks for ED can come with age, with the percentage of men suffering from
this disease increasing by the time a patient is above 40 years old. Smoking,
Page 1
drinking, and drugs also aggravate the situation, damaging nerves, blood
vessels, or even the brain.
Excessive cycling may also lead to erectile dysfunction. Trauma to the genital
area for a prolonged time is also a risk factor. Apart from physical trauma on
the penis, the testosterone-producing capability of the testes also take a hit.
Autonomic
o Sympathetic nerves from T11-L2 - Sympathetic activity
causes ejaculation and detumescence (loss of erection)
o Parasympathetic from S2-4, form the pelvic plexus which
causes erection

Somatic
Additional notes: Based on researchers, excessive masturbation can
o Somatosensory information travels via the pudendal nerves
cause ED. Kaya boys, hinay lang.
o Efferent innervation of the ischiocavernosus and
Question: Can exercise cause ED?
bulbocavernosus muscles of the penis
Yes, as discussed earlier, excessive cycling (cycling is a form of
Central
exercise) can lead to ED
o Medial preoptic area and paraventricular nucleus (PVN) in
the hypothalamus are important centers for sexual function
Discuss the biological and physiological mechanisms involved in the and penile erection
normal process of erection. What are the defects and/or abnormalities
resulting to ED?

Penile erection is a complex process combining psychological stimuli,

neurologic event, smooth muscle relaxation, arterial dilation and venous
compression ultimately resulting to a state of the penis that is filled with
blood and becomes rigid.

2 Mechanisms:

Reflex erection
o Achieved by directly touching the penile shaft
o Uses the peripheral nerves and the lower parts of the spinal
cord
Psychogenic erection
o achieved by erotic or emotional stimuli
o uses the limbic system of the brain

What is innervation of Erection?

BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E Page 2

MECHANISM OF ERECTION OVERVIEW OF BIOCHEMICAL EVENTS

Neuroendocrine signals from the brain, created by audiovisual, tactile or olfactory stimuli

Signals are relayed via the cavernosal nerve to the erectile tissue of the copora
cavernosa, activating the veno-occlusive mechanism

This triggers increased arterial blood flow into sinusoidal spaces with relaxation of
cavernosal smooth muscle, and opening of the vascular space

Compressing the subtunical venous plexuses, decreasing venous outflow

Both spongiosus and cavernosus are surrounded by tunica albuginea, which consist of
outer longitudinal and inner circular layers. The sliding of 2 layers over each other
during engorgement lead to occlusion of emissary veins

Rising intracavernosal pressure and contraction of the ischiocavernosus muscles

produces a rigid erection

BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E Page 3

Why do diabetics (DM) have higher risk of developing ED? Explain Maillard
reaction and its consequence of adduct formation.

Men who have diabetes are two to three times more likely to have ED than
men who do not have diabetes. Among men with ED, those with diabetes
may experience the problem as much as 10 to 15 years earlier than men
without diabetes. Research suggests that ED may be an early marker of
diabetes, particularly in men ages 45 and younger.

The chemical reactions leading to the non-enzymatic glycation of the What is Viagra? Discuss why its indicated in ED and show its mechanism
protein is termed as Maillard reaction or advanced glycation. Glycation is of action.
the formation of complexes between sugars and amino acids of proteins Viagra (Sildenafil), a prescription medicine used to treat ED
that cause toughening and discoloration of food during the cooking process enhances the effect of NO by inhibiting phosphodiesterase type 5 (PDE5).
and after prolonged storage. Sustained hyperglycemia results to glycation of PDE5 is responsible for the degradation of cGMP in the corpus cavernosum.
the proteins at the amino group of the lysine residue. Reducing sugars, such Sildenafil has no direct relaxant effect on isolated human corpus
as glucose, react non-enzymatically with the free amino groups of proteins cavernosum. When sexual stimulation causes local release of NO, inhibition
to initiate advanced glycation, resulting in the formation of reversible Schiff of PDE5 by sildenafil causes increased levels of cGMP in the corpus
bases, which by intermolecular rearrangement are converted into stable, cavernosum, resulting in smooth muscle relaxation and inflow of blood to
covalently-bonded Amadori products. When large amounts of Amadori the corpus cavernosum.
products are accumulated, they undergo further rearrangement and cross-
linkage to form Advanced Glycated End Products (AGEs). The rate of
glycation is increased in a hyperglycemic condition. Furthermore, some of Give other drugs currently available other than Viagra in treating ED.
the AGEs form covalent crosslinks with adjacent protein strands. This
crosslinking stiffens tissues which were formerly flexible or elastic. Such ONSET DURATION
modified species bind to and activate the receptors for advanced glycation
end products (RAGE) present on the surface of multiple cell types, including Viagra (sildenafil) 30-60 minutes 4 hours
endothelial cells (ECs). AGERAGE interactions result in EC dysfunction as
AGEs block nitric oxide activity in the endothelium, and appear to play an Levitra (vardenafil) 30-60 minutes 8 hours
important pathophysiological role in several diseases, including type 1 and Staxyn (vardenafil) 15 minutes 6 hours
type 2 diabetes.
Cialis (tadalafil) 30-60 minutes 8 hours

Stendra (avanafil) 15-30 minutes 6 hours

BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E Page 4

 Phosphodiesterase type 5 (PDE-5) inhibitors such as Viagra, Levitra,
and Cialis work by increasing the blood flow to the penis when sexually
aroused. They are all similar in effect but onset of action and duration are
different. They may only be taken once a day.
Cialis - can be taken with or without food and should be taken about 30 to
60 minutes before sexual activity. It can stay active in your body for up to
eight hours.
Levitra - patient should take it once a day on an empty stomach. It works
best if you take it about 30 to 60 minutes before sex, and it can stay active
for as long as eight hours.
Viagra - works best on an empty stomach. High-fat foods can interfere with
Viagra, resulting in it taking longer to work. Take the pill about 30 to 60
minutes before sex. It usually stays active for about four to five hours.
However, it can sometimes last eight hours or more.
enormous chain reaction of free radicals that quickly wreaks havoc on living
tissue and eventually leads to tissue injury.
FREE RADICAL FORMATION
Initiation phase: there is production of free radicals from a bonded
molecule. The bond between them is destroyed via light, heat or sometimes
through oxidation processes wherein there will be a loss of electron.
Propagation phase: there is stealing of electrons from a stable molecule for
a free radical to stabilize itself then it will now turn the once stable molecule
into a free radical and the same thing happens.
Termination phase: there is union of one free radical with another free
radical, thus, forming a stable molecule again.

Stendra - 100 or 200 mg is taken as early as approximately 15 minutes

before sexual activity, while Stendra 50 mg is taken approximately 30
minutes before sexual activity.

Staxyn - is an orally disintegrating tablet and should be taken an hour

before sexual activity.

TOPIC: ANTIOXIDANTS AND GLUTATHIONE

What are free radicals? How are they formed and what are their sources?

A free radical is an atom or molecule that has an unpaired electron

in its outer orbit which makes them highly reactive. In order to stabilize
themselves, free radicals steal an electron from a neighboring molecule.

Once a free radical forms and it succeeds in gaining another

electron from a nearby molecule, it leaves its victim short an electron and
has now made this new molecule a free radical, which will in turn, try and
steal an electron as well. The result is what we call a free radical cascade, an
BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E Page 5

From oxygen, superoxide can be formed through transfer of 1
electron or from enzymes xanthine oxidase and cytochrome P450.
Then, superoxide can be converted to hydrogen peroxide by adding
2 electrons or by the enzyme, superoxide dismutase.
Through transfer of 3 electrons, enzymes such as catalase and GSH
and through Fenton Reaction,
Hydroxyl radical can be formed then four electrons can form a
water molecule which is now stable. You can also form a hydroxyl
ion from water via radiolysis.
What are the deleterious effects of free radicals on the body?
When produced in excess, free radicals and oxidants generate a
phenomenon called oxidative stress, a deleterious process that can seriously
alter the cell membranes and other structures such as proteins, lipids,
lipoproteins, and deoxyribonucleic acid (DNA)
Hydroxyl radical and peroxynitrite in excess can damage cell
membranes and lipoproteins by a process called lipid peroxidation. This
reaction leads to the formation of malondialdehyde (MDA) and conjugated
diene compounds, which are cytotoxic and mutagenic.
Lipid peroxidation occurs by a radical chain reaction, i.e. once
started, it spreads rapidly and affects a great number of lipid molecules.
Proteins may also be damaged by ROS/RNS, leading to structural changes
BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E
and loss of enzyme activity. Oxidative damage to DNA leads to the
formation of different oxidative DNA lesions which can cause mutations.
Oxidative stress can induce a variety of chronic and degenerative
diseases as well as the aging process and some acute pathologies (trauma,
stroke).
Cancer - Cancer initiation and promotion are associated with
chromosomal defects and oncogene activation induced by free radicals. A
common form of damage is the formation of hydroxylated bases of DNA,
which are considered an important event in chemical carcinogenesis. This
adduct formation interferes with normal cell growth by causing genetic
mutations and altering normal gene transcription.
Lipid peroxidation in cancer causes leukemia, breast, ovary,
rectum cancers among elderly people.
Radical damage to DNA in germ-line cells in ovaries and testes can
lead to heritable mutations; in somatic cells the result may be initiation of
cancer.
Cardiovascular Diseases - Chemical modification of the proteins or
lipids in plasma low density lipoprotein (LDL) leads to abnormal LDL that is
not recognized by the liver LDL receptors, and so is not cleared by the liver.
Autoimmune Diseases - Chemical modification of amino acids in
proteins, either by direct radical action or as a result of reaction with the
products of radical-induced lipid peroxidation, leads to proteins that are
recognized as non-self by the immune system. The resultant antibodies will
also cross-react with normal tissue proteins, so initiating autoimmune
disease.
What are anti-oxidants, What are their general functions?
An antioxidant is a molecule stable enough to donate an electron to
a rampaging free radical and neutralize it, thus reducing its capacity to
damage. They can safely interact with free radicals and terminate the chain
Page 6
reaction before vital molecules are damaged. Some of such antioxidants,
including glutathione, ubiquinol, and uric acid, are produced during normal
metabolism in the body. Other lighter antioxidants are found in the diet.
Although there are several enzymes system within the body that scavenge
free radicals, the principle micronutrient (vitamins) antioxidants are vitamin
E (-tocopherol), vitamin C (ascorbic acid), and B-carotene. The body cannot
manufacture these micronutrients, so they must be supplied in the diet.
The first line of defense is the preventive antioxidants, which
suppress the formation of free radicals.
The second line of defense is the antioxidants that scavenge the
active radicals to suppress chain initiation and/or break the chain
propagation reactions. These are composed of hydrophilic and lipophilic
antioxidants
Hydrophilic antioxidants - Vitamin C, uric acid, bilirubin, albumin,
and thiols
Lipophilic antioxidants - Vitamin E and ubiquinol
Vitamin E accepted as the most potent radical scavenging lipophilic
antioxidant
A. Vitamin E together with a reactive free radical (a hydroxyl ion) can
produce a less reactive TO and a stable water.
BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E
B. Vitamin C can give electron to a free radical yields off a stable Asc
and a stable ROOH.
C. You can also convert a less reactive TO by using Vitamin C into
Vitamin E and a stable Asc.
The third line of defense is the repair and de novo antioxidants. The
proteolytic enzymes, proteinases, proteases, and peptidases, present in the
cytosol and in the mitochondria of mammalian cells, recognize, degrade,
and remove oxidatively modified proteins and prevent the accumulation of
oxidized proteins.
NOTE: I didnt include question number 4 in the manual as Maam
Alcantara said it has nothing to do with the topic
What are flavonoids? (Will explain only its antioxidant properties: As said
by Maam Alcantara)
Flavonoids are a large family of polyphenolic compounds
synthesized by plants that have a common chemical structure. They are
collectively known as Vitamin P or Citrin. They are secondary metabolites
characterized by a flavan nucleus and a C6-C8-C6 carbon skeleton. The basic
structural feature of flavonoid is the 2-phenyl-benzo--pyrane nucleus
consisting of two benzene rings (A and B) linked through a heterocyclic
pyran ring (C).
Flavonoids are powerful antioxidants against free radicals and are
described as free-radical scavengers. This activity is attributed to their
hydrogen- donating ability. The phenolic groups of flavonoids serve as a
source of a readily available H atoms such that the subsequent radicals
produced can be delocalized over the flavonoid structure. Free radical
scavenging capacity is primarily attributed to high reactivities of hydroxyl
substituents that participate in the reaction.
Flavonoid(OH) + R > flavonoid(O) + RH
Page 7
 Flavonoids inhibit lipid peroxidation in vitro at an early stage by
acting as scavengers of superoxide anion and hydroxyl radicals.
What is glutathione? What is its chemical composition and metabolism?
Glutathione (GSH) is a tripeptide that contains an unusual peptide linkage
between the amine group of cysteine and the carboxyl group of a
glutamate side-chain. It is an antioxidant, preventing damage to
important cellular components caused by reactive oxygen species such as
free radicals and peroxides.
Thiol groups are reducing agents. Glutathione reduces disulfide formed
within cytoplasmic proteins to cysteines by serving as an electron donor. In
the process, glutathione is converted to its oxidized form Glutathione
disulfide (GSSG), also called L- Glutathione.
Once oxidized, glutathione can be reduced back by glutathione
reductase, using NADPH as an electron donor.
CHEMICAL COMPOSITION
Glutathione is not an essential nutrient and can be synthesized in the body
from the amino acids L-cysteine, L-glutamic acid, and glycine. The sulfhydryl
(thiol) group (SH) of cysteine serves as a proton donor and is responsible for
the biological activity of glutathione. Provision of this amino acid is the rate-
limiting factor in glutathione synthesis by the cells, since cysteine is
relatively rare in foodstuffs.
BIOSYNTHESIS
Glutathione is synthesized in 2 ATP-dependent steps:
First, gamma-glutamylcysteine is synthesized from glutamate and cysteine
via the enzyme gamma-glutamylcysteine synthetase (a.k.a. glutamate
cysteine ligase, GCL). This reaction is the rate-limiting step in glutathione
synthesis.
BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E
Second, glycine is added to the C-terminal of gamma-glutamylcysteine via
the enzyme glutathione synthetase.
Discuss the role of GSH in infection, immunity, detoxification,
cardiovascular diseases, pulmonary diseases, Parkinsons disease and
aging
Infection - AIDS and glutathione depletion are closely related. People
with AIDS naturally have low levels GSH. Glutathione levels rapidly decrease
with AIDS since the immune cells use it to fight off oxidative stress caused
by the infection. The HIV virus directly attacks the white blood cells who, in
turn, use its glutathione to defend itself and the body.
Research suggests that an increase of intracellular GSH inhibits HIV
replication
Immunity Lymphocytes depend on glutathione for their proper function
and replication. Glutathione activates and proliferates lymphocytes by
donating cysteine to leukotrienes during eicosanoid synthesis via the
enzyme glutathione-s-transferase.
Detoxification - Detoxifies or helps remove toxins when they enter the body
and subsequently the individual cells in the organs.
Cardiovascular Diseases - Studies have shown that total glutathione
concentrations were lower in all cardiovascular disease cases than in control
subjects.
GSH protects your heart and brain in the event of an attack. GSH
diminishes damage to oxygen-deprived tissue during and after the
attack.
GSH protects against lipid peroxidation the process that turns
cholesterol rancid. This process causes cholesterol deposits to stick
to the artery walls.
Page 8
 GSH has been shown to prevent and even reverse hardening of the
endothelial the elastic inner lining a condition known as
arteriosclerosis.
Pulmonary Disease - Inflammatory lung diseases are characterized by
chronic inflammation and oxidant/antioxidant imbalance, a major cause of
cell damage. Glutathione (GSH), is a vital intra- and extracellular protective
antioxidant against oxidative stress, which plays a key role in the control of
pro-inflammatory processes in the lungs
Parkinsons Disease - Glutathione has the unique ability to make certain
areas of the brain more sensitive to dopamine, so that even though
dopamine is decreased, it nevertheless becomes more effective.
In addition, GSH protects the brain from free radical damage. Complexing
with NO helps in promoting growth and protection of dopamine-producing
cells in the substancia nigra, preventing it from being damaged by oxidative
stress.
Aging - Studies suggest that elevated GSH levels give elderly individuals a
physical, psychological and sociological advantage over those with lower
levels. Studies also found that higher glutathione levels corresponded to
lessened effects of aging and better general health.
Discuss its role as a whitening agent
Direct inactivation of the enzyme tyrosinase by binding with the
copper-containing active site of the enzyme
Mediating the switch mechanism from eumelanin to phaeomelanin
production
Quenching of free radicals and peroxides that contribute to
tyrosinase activation and melanin formation
Modulation of depigmenting abilities of melanocytotoxic agents.
BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E
TOPIC: APOPTOSIS AND CELL NECROSIS
1. Define Apoptosis
Apoptosis is a pathway of cell death in which cells activate enzymes that
degrade the cells own nuclear DNA and nuclear and cytoplasmic proteins.It
is an orderly cell death that results in disassembly and phagocytosis of the
cell before any leakage of its contents occurs, and neighboring cells usually
remain healthy. This is initiated by the activation of a family of proteases
called caspases.
Causes of Apoptosis
Apoptosis occurs in many normal situations and serves to eliminate
potentially harmful cells and cells that have outlived their usefulness. It also
occurs as a pathologic event when cells are damaged beyond repair,
especially when the damage affects the cells DNA or proteins; in these
situations, the irreparably damaged cell is eliminated.
Apoptosis in Physiologic Situations
Death by apoptosis is a normal phenomenon that serves to eliminate cells
that are no longer needed and to maintain a constant number of cells of
various types in tissues.
The programmed destruction of cells during embryogenesis.
Normal development is associated with the death of some cells and the
appearance of new cells and tissues.
Involution of hormone-dependent tissues upon hormone deprivation.
During the endometrial cell breakdown during the menstrual cycle, and
regression of the lactating breast after weaning
Page 9
 2. Differentiate Apoptosis from Necrosis
Apoptosis
Introduction Apoptosis, or programmed cell
death, is a form of cell death that is
generally triggered by normal,
healthy processes in the body.
Natural Yes
Effects Usually beneficial. Only
abnormal when cellular processes
that keep the body in balance
cause too many cell deaths or too
few.
Process Membrane blebbing, shrinkage
of cell, nuclear collapse (nuclear
fragmentation, chromatin
condensation, chromosomal
DNA fragmentation), apoptopic
body formation. Then, engulf by
white blood cells.
Symptoms Usually no noticeable symptoms
related to the process.
Causes Self-generated signals in a cell.
Generally natural part of life, the
continuation of the cellular cycle
initiated by mitosis.
Medical Very rarely needs treatment.
Treatment
BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E
Necrosis
Necrosis is the premature
death of cells and living
tissue. Though necrosis is
being researched as a
possible form of programmed
cell death, it is considered an
"unprogrammed" cell death
process at this time.
Caused by factors external
to the cell or tissue, such as
infection, toxins, or trauma.
Always detrimental
Membrane disruption,
respiratory poisons and
hypoxia which cause ATP
depletion, metabolic collapse,
cell swelling and rupture
leading to inflammation.
Inflammation, decreasing
blood flow at affected site,
tissue death
Bacterial or fungal
infections, denatured
proteins that impede
circulation, fungal and
mycobacterial infections,
pancreatitis, deposits of
antigens and antibodies
combined with fibrin.
Always requires medical
treatment. Untreated
necrosis is dangerous and can
lead to death.
3. What are the different cellular features of both apoptosis and
necrosis?
FEATURE APOPTOSIS NECROSIS
CELL SIZE reduced (shrinkage) enlarged (swelling)
NUCLEUS Fragmentation into Pyknosis
nucleosome size fragments karyorrhexis
karyolysis
PLASMA MEMBRANE Intact; altered structure, disrupted
especially orientation of
lipids
CELLULAR CONTENTS Intact; may be released in Enzymatic digestion;
apoptotic bodies may leak out of cell
ADJACENT INFLAMMATION No Frequent
PHYSIOLOGIC OR Often physiologic; means Invariably pathologic
PATHOLOGIC ROLE of eliminating unwanted
(culmination of
cells; may be
irreversible cell injury)
pathologic after some
forms of cell injury,
especially DNA and
protein damage
4. What are the different internal and external signals that define the
apoptotic phenomenon? How does apoptosis happen inside the cells?
In the extrinsic pathway, signal molecules known as ligands, which are
released by other cells, bind to transmembrane death receptors on the
target cell to induce apoptosis
The intrinsic pathway is triggered by cellular stress, specifically
mitochondrial stress caused by factors such as DNA damage and heat shock.
Page 10
Upon receiving the stress signal, the proapoptotic proteins in the cytoplasm,
BAX and BID, bind to the outer membrane of the mitochondria to signal the
release of the internal content.
5. What is the role of apoptosis in:
Embryogenesis - Programmed cell death plays an important role in the
processes of gamete maturation as well as in embryo development,
contributing to the appropriate formation of various organs and structures.
Cell death for example, helps sculpt hands and feet during embryonic
development; they start out as spade like structures, and the individual
digits separate only as digits separate only as the cells between them die.
Development and differentiation of tissues - Specific populations of cells
are eliminated by programmed cell death at different stages of
embryogenesis and also in adult tissues. The morphogenesis of organs
typically involves the coordination of several cellular processes including
migration, proliferation, apoptosis and changes in cell shape or polarity
AIDS-The mechanisms of HIV-1-mediated cell death which are relevant in
vivo are unclear at present. However, in vitro explorations on the cytopathic
effects of HIV-1 have yielded a wealth of potential triggering events, and
signaling and effector pathways leading to apoptosis
6. What are the different laboratory indices that will define cells
undergoing apoptosis?
Apoptosis can be detected in the laboratory through these four events:
Plasma membrane alterations
Mitochondrial changes
Changes in the cytoplasm
DNA modifications
Plasma membrane alterations During early stages of apoptosis, certain
characteristic changes appear on the membrane.
BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E
Mitochondrial changes There is a disruption in the function of the
mitochondria during apoptosis. This disruption causes changes in the
membrane permeability which allow some molecules to pass through/leak.
Changes in the cytoplasm A distinctive feature of the early stages of
apoptosis is the activation of caspase enzymes. Activated caspases cleaves a
dye conjugated DEVD peptide. The dye is free to bind to a DNA producing a
fluorescent signal.
TOPIC: TELOMERASE AGING AND CANCER
How does a chromosome look like? What makes up the centromere and
telomeres of a chromosome?
PARTS OF A CHROMOSOME
Heterochromatin usually contain junk DNA. Junk DNA are spacers which
do not code for anything. This type of chromatin are packed tightly
Euchromatin usually contain active genes (transcriptionally active). They
are lightly stained because they are lightly packaged (which enables them to
be accessed easier for transcription compared to compact structures).
Centromere - Condensed regions within the chromosome that are
responsible for the accurate segregation of the replicated chromosomes
during mitosis and meiosis
Centromeres are attached to microtubules (proteins that can pull
chromosomes toward opposite ends of each cell prior cell division).
Centromeres are made up of protein A (CENP-A) which is important for the
localization and assembly of kinetochore proteins.
Additional notes: Kinetochore is a complex of proteins that forms each
centromere and serves as the attachment point for spindle fibers that
will separate the sister chromatids during mitosis.
Page 11
All human centromeres has presence and abundance of AT-rich satellite Laying down of primer by RNA primase for leading strand and laying
family, known as alpha satellite. Centromeres mediates the union of two down of several primers by RNA primase for lagging strand
chromatids and also play a role in kinetochore assembly.
B. Elongation
Telomeres - complexes of noncoding DNA plus proteins (collectively known
Leading strand
as shelterin) located at the ends of linear chromosomes
Addition of nucleotides by DNA polymerase delta (along replication
Telomeres maintain the structural integrity of the chromosome and protect
fork)
the core or the inside of the DNA molecule which contains the genetic code
Lagging strand
The characteristic repeat sequence found in telomeric region is TTAGGG

Addition of nucleotides by DNA polymerase delta (away from

Review briefly the process of replication of a linear chromosome. What
replication fork)
accounts for the natural shortening of the lagging strand?
Formation of Okazaki fragments
Prokaryote Eukaryote
Replacement of RNA primers with DNA polymerase alpha
Semi-conservative Semi-conservative
Filling in the gaps by DNA ligase.
Primer Dependent Primer-Dependent

Single origin of replication Multiple origin of replication

C. Termination unclear mechanism
DNA Pol III creates both leading DNA pol alpha and DNA pol delta
and lagging strand creates lagging and leading strand
respectively

Supercoiling Telomere shortening (kasi linear

and supercoiling)

EUKARYOTIC REPLICATION

A. Initiation

Unwinding of DNA by helicase

Binding of Single stranded binding proteins

BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E Page 12

LAGGING STRAND FORMATION
Unwinding by helicase
Annealing of primers by primase
Elongation by DNA Pol delta
Removal and replacement of primase by DNA Pol alpha
Ligase activity
Note: since OH is a requirement in creating a phosphodiester bond among
nucleotides, removal of the primer at the 5' end of the lagging strand leaves no OH
This results to the inability of replacing primers with DNA nucleotides with DNA
alpha
3' overhand (shortening of lagging strand)
What is cellular senescence? Expound.
Cellular Senescence is formally described as a process that limited the
proliferation or growth of normal human cells in culture. It denotes a stable
and long-term loss of proliferative capacity, despite continued viability and
metabolic activity disrupting metabolism resulting in deterioration and
death. Also refers to essentially irreversible growth arrest that occurs when
cells that can divide encounter oncogenic stress.
BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E
MECHANISM OF CELLULAR SENESCENCE
Telomeric Shortening
During replication, when all the primers have been taken out, the primer
located at the DNA ends is not replaced with actual DNA because there is no
presence of 3-OH, which serves as the attachment site for DNA synthesis.
When ligase connects the adjacent Okazaki fragments together, the last one
has no replacement therefore, it is now SHORTER compared to the original
one (3OVERHAND). Everytime the DNA undergoes replication; it becomes
shorter until nothing is left - SENESCENT
Accumulation of Damage
The mutations caused by many factors would sum up, and this leads to
formation of double strand breaks (DSBs) and single strand breaks (SSBs) in
the DNA strand. These DNA damage would trigger a DNA damage response
(DDR) and activate the p53 pathway and the cell would enter the senescent
phase in order to repair the damage.
Additional notes: Just in case that theyll ask, cellular senescence was
first observed by Leonard Hayflick
Hayflick limit number of times a normal human cell population will
divide until cell division stops.
Glycation
In this mechanism, there is involvement of carbohydrate mainly glucose that
undergoes non-enzymatic fusion through covalent bonds with proteins and
nucleic acids.
The first step is glycation itself where CHON, nucleic acids and glucose form
bonds. This ends up forming Advanced Glycation End products (AGE) that
interferes with natural processes of cell. These AGE damages cells by
enhancing oxidative stress by reacting with substances and it releases
radicals in the process.
Page 13
Cross-linking Deletions in the mitochondrial DNA (mtDNA) during replicative
senescence
Changes of DNA metabolism in association with cellular aging may be
attributed primarily to the crosslinking of core histone subunits. Further Secretes cytokines and chemokines (trigger a variety of cellular
speculation awaits substantive data showing increased histone crosslinking responses)
in senescent cells and also what crosslinked histones in various DNA
Senescence and Cancer
metabolisms may imply.
Senescence has TUMOR SUPPRESSING mechanism, limiting the
tTGase is a senescent marker (enzyme responsible for cross linking)
replicative potential of pre-neoplastic cells (altered gene expression, growth
Mitochondrial DNA (mtDNA) Damage Theory arrest and apoptosis).

In normal conditions, the oxygen is reduced to produce water; however,

oxygen is instead prematurely and incompletely reduced to give the
superoxide radicals and reactive oxygen species (ROS). These are highly
reactive molecules and cause mutations or damage to the mtDNA. When a
new mtDNA mutation arises in a cell, a mixed population of mutant and
normal mtDNA molecules is generated, known as heteroplasmy. As the cell
replicates, the mutant and normal mtDNAs are randomly distributed into
daughter cells. As the percentage of mutant mtDNAs increases, the energy-
generating capacity of the cell or tissue declines. Since each tissue has a
characteristic mitochondrial energy requirement, its "bioenergetic
threshold" is not met due to decline in mitochondrial energy producing
capacity. These results to cells that weaken due insufficient energy supply
which manifest first on tissues highly dependent on ATP like neurons.
Properties of a cell during senescent phase
Bigger, has more diverse morphology types
What is the enzyme telomerase? Discuss telomerase as to the following
aspects.
STRUCTURE (protein and RNA components, repeat motifs)
The human telomerase enzyme complex consists of two molecules each of
human telomerase reverse transcriptase (TERT), telomerase RNA (TR or
TERC), and dyskerin (DKC1).
The genes of telomerase subunits, which include TERT, TERC, DKC1 and
TEP1, are located on different chromosomes. TERT polypeptide folds with
(and carries) TERC.
TERT has a 'mitten' structure that allows it to wrap around the chromosome
to add single-stranded telomere repeats. TERT is a reverse transcriptase,
which is a class of enzyme that creates single-stranded DNA using single-
stranded RNA as a template.

Some cells have smaller cellular density due to a more sensitive cell
to cell inhibition (contact inhibition)

Lysosomes increase in number and size

Aging will increase digestion of cells organelles

BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E Page 14

SPECIFIC CELL POPULATIONS
Epidermal skin cells, follicular hair cells
Pluripotent stem cells such as embryonic stem cells
Multipotent stem cells such as neural stem cells
Cancer stem cells
MECHANISM OF TELOMERIC LENGTHENING
When a chromosome is replicated, the 3' hanging parental fragment
containing the G-rich sequence GGGTTA (in humans) is recognized by the
telomerase complex.
Using its own RNA as a complementary primer, the telomerase mimics
reverse transcriptase and elongates the hanging DNA fragment in the 5' to
3' direction, moving down the growing strand until it is a telomere several
thousand bases longer.
Replication of the lagging strand can now be completed using the telomeric
extensions as a template for synthesis using DNA polymerase.
With this mechanism, the 3' end of each telomere is still slightly longer than
the complementary 5' end. Other telomere-associated proteins move in to
loop the protruding end back on itself, protecting it from degrading
enzymes and other mechanisms for repairing broken DNA.
By using TERC, TERT can add a six-nucleotide repeating sequence, 5'-
TTAGGG (in vertebrates, the sequence differs in other organisms) to the 3'
strand of chromosomes. These TTAGGG repeats (with their various protein
binding partners) are called telomeres.
The template region of TERC is 3'-CAAUCCCAAUC-5'.
SHORTCUT:
Telomerase can bind the first few nucleotides of the template to the last
telomere sequence on the chromosome >> add a new telomere repeat (5'-
BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E
GGTTAG-3') sequence >> let go >> realign the new 3'-end of telomere to the
template, and repeat the process. Telomerase reverses telomere
shortening.
MECHANISM OF TELOMERIC SEALING
At the very distal end of the telomere is a 300 bp single-stranded portion,
which forms the T-Loop. This loop is analogous to a knot, which stabilizes
the telomere, preventing the telomere ends from being recognized as break
points by the DNA repair machinery.
Should non-homologous end joining occur at the telomeric ends,
chromosomal fusion will result.
The T-loop is held together by several proteins, the most notable ones being
TRF1, TRF2, POT1, TIN1, and TIN2, collectively referred to as the shelterin
complex. In humans, the shelterin complex consists of six proteins identified
as TRF1, TRF2, TIN2, POT1, TPP1, and RAP1.
This event plays a protective role as it sequesters the overhang
terminal inside the double strand. It protects the chromosome from fusion,
allow as cell to distinguish chromosomes ends from sites of DNA damage
and it helps to regulate the length of telomere.
What prevents the telomerase from over-extending the ends of a linear
chromosome? Discuss the role of telomere binding proteins in the
regulation of telomerase function.
TRF1 (telomeric repeat binding factor 1)
Expressed ubiquitously throughout the cell cycle
Binds to TTAGGG repeat as a homodimer (at t-loops) with great
specificity
Functions in cis to inhibit telomerase-dependent elongation
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 Participates in regulation of the mitotic spindle
Negative regulator of telomere length (telomerase-dependent
pathway)
TRF2 (telomeric repeat binding factor 2)
Expressed ubiquitously throughout the cell cycle
Binds to TTAGGG repeat as homodimer with great specificity
Localizes to t-loops, involved in their formation
C-terminal domains homologous to MYB family of proto-oncogenes
Might be involved in inhibition of replication fork
Stabilizes the G-rich strand overhang and inhibits telomere-telomere
fusions
TRF2-negative telomeres are recognized as damaged DNA
Negative regulator of telomere length; TRF2 overexpression in
somatic cells = telomere shortening
TRF2 inhibition causes apoptosis and non-homologous end joining
(NHEJ) of telomeres
Promotes binding of hRAP1, a telomere associated protein
hRAP1
Negative regulator, in cis, of telomere length
C-terminus mediated interaction with TRF2
Functions in determining relative telomere length
TIN2 (TRF1-interacting nuclear factor 2)
Regulates telomere length via NH2-terminus mediated binding to
TRF1
BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E
Mutant TIN2 lacking NH2-terminus leads to elongated telomeres
Promotes TRF1-dependent pairing of telomere repeat.
STAGES OF TELOMERE SHORTENING
M1 stage Normal cells in culture replicate until they reach a
discrete point at which population growth ceases. This is caused by
the shortening of few telomeres in to a size.
M2 stage Also called as the crisis stage. M1 stage can be bypassed
in vitro by abrogation of the function of the p53 and pRB (which are
human tumor suppressor genes). The cells can then continue to
replicate until the telomeres become extremely short and becomes
prone to end-to-end fusions and chromosome breakage fusion cycles
Additional notes: Telomere length negatively correlates with age.
Telomere length in humans seems to decrease at a rate of 24.8-27.7
base pairs per year.
Certain lifestyle factors such as smoking (due to oxidative stress),
obesity (deregulated production of adipocytokines), lack of exercise,
and consumption of unhealthy diet can increase the pace of telomere
shortening, leading to illness and/or premature death.
Page 16
Can telomerase be used as an agent to extend cellular lifespan? YES
A study conducted by Bodnar and colleagues involved two groups of
cells. One was hTRT (-) and the other was hTRT (+). Activation of telomerase
via the incorporation of its catalytic subunit [hTRT (+)] resulted to an
increase in the number of telomeres and population doubling. This is
accompanied by fewer -galactosidase staining cells which signifies lesser
cells approaching senescence compared to hTRT (-) groups.
Additional notes: What are the benefits of telomerase lengthening in
comparison to other cell cultures?
Primary cell lines susceptible to replicative senescence
Continuous cell lines susceptible to mutations (can live long though
kasi maintained sya in a continuous culture)
Telomerase extended cells not prone to mutations plus lives long!
Is it possible to revert oid cells into young cell again? YES
Jaskelioff and collegueas confirmed through their study that telomerase
reactivation reverses tissue degeneration in aged telomerase deficient mice.
Artificially aged mice exhibited tissue atrophy, organ system failure,
progressive restriction of neurogenesis and remyelination, decreased self-
renewal ability, decreased fecundity and survival. Treatment by hTERT re-
activation resulted to resumption of cell proliferation, recovery from tissue
atrophy, improved brain function, and increased fecundity. hTERT
reactivation also ameliorated the decrease of survival of mice.
These findings show that aging induced by telomere loss can be
reversed in a broad range of tissues and cell types, including neuronal
function.
BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E
Can anti telomerase agents be used to treat cancer cells?
Anti-sense oligodeoxynucleotides (AS-ODN)
The drugs consists of short stretches of DNA that are complementary to a
target RNA. The mechanism of action for most applications is to hybridize to
their complementary RNA by Watson-Crick base pairing and inhibit the
translation of the RNA by passive or active mechanism.
Passive mechanism occurs by competitive binding of ODN to the
RNA
Active mechanism cleavage of the target hTR post-hybridization
by RNase H
Hammerhead Ribozymes
They are small catalytic RNA molecules which possess specific endonuclease
activity which can catalyze self-cleavage reaction.
Dominant Negative hTERT
Dominant negative (DN)-hTERT constructs are catalytically inactive hTERT
mutants which can potentially inhibit telomerase activity from maintaining
telomeres by binding and sequestering hTR.
Reverse Transcriptase Inhibitors
Reverse transcriptase inhibitors (RTI) are a class of molecules which
hinder the proper functioning of reverse transcriptase enzyme. They usually
act by competing with the endogenous nucleic acids to integrate into the
DNA and hence subsequently, they interfere with the chain elongation
function of reverse transcriptase.
Page 17
TOPIC: DEPRESSION Dopamine
Increased level in the frontal lobe of the brain contributes to the incoherent
Difference between Hormones and Neurotransmitters and distruptive thought processes that are characteristic of schizophrenia.
Excessive levels of DA cause our thinking to be excited, energized, then
Neurotransmitter Hormone suspicious and paranoid as we are hyperstimulated by our environment.
Epinephrine (adrenaline)
Mechanism of release Through synapse Bloodstream Epinephrine is another type of adrenaline which also brings out the arousal
of extreme emotions such as fear, anger, or amusement in reaction with the
different instances a person is involved in. People commonly call emotions
Secretory organs Vesicles in the synaptic Endocrine glands that are related to such as adrenaline rush.
bulb
Norepinephrine
Elevated levels is a contributor to anxiety. During conditions of stress, brain
NEp turnover is increased. Increased levels of NEp leads to alertness and
Duration of response Quick , instantenous Longer, prolonged
mood elevation, and increased sexual interest. Low levels are linked to lack
and effect effect
of energy, focus and motivation. It also contributes to depression, loss of
alertness, and poor memory.

Associated with Nervous System Endocrine System Serotonin

Hormones/neurotransmitters playing significant roles in a persons result in depressed moods, anxiety, panic attacks, obsessions or
emotions and its imbalances which can result to depression.
These hormones include Estrogen, Progesterone, Testosterone and
Oxytocin. The following neurotransmitters also affecting human emotion
are as follows: Norepinephrine and Epinephrine, Serotonin, GABA,
Dopamine, and Acetylcholine.
GABA (gamma-aminobutyric acid)
Excessive GABA levels result in excessive relaxation and sedation to the
point of normal reactions are impaired. Insufficient GABA on the other
hand, results in the brain being overstimulated. People with too little GABA
tend to suffer anxiety disorders. Low levels of GABA are associated with
bipolar disorders, mania, poor impulse control, epilepsy, and seizure
disorders.
Excessive amounts can cause sedation, a decrease in sexual drive, a sense of
well being, bliss and being one with the universe. Decreased levels can
compulsions, feeling tense and irritable, angry, and decreased libido
Estrogen
Estrogens effect on mood is due to its ability to increase serotonin, and
beta-endorphins which are associated with positive mood states. Decreased
levels of estrogen in women may lead to develop symptoms of depression,
anxiety, and mood swings. Excessive levels on the other hand also causes
anxiety, and irritability.
Progesterone
Progesterone is another hormone which serves as a key element to
reproductive success in women. This however, has greater influence to
brain while its effect to emotion depends on its balance with estrogen.

BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E Page 18

While this hormone is responsible in promoting good sleep and improving
libido, imbalance of such can lead to insomnia, headaches, and anxiety.
Testosterone
Proper balance of this hormone is needed to increase production and
motility of sperm. Otherwise, imbalance of such would result to decreased
sex drive in men that are sometimes brought about by erectile dysfunction
as well as fatigue and low sperm production. This is also when men suffer
from fertility issues and experience a hard time achieving conception with
the partner.
Oxytocin
It is responsible for reproductive function in humans and is released during
sexual orgasm in couples. Some call this as love hormone while this also
helps build good interpersonal relationship in couples as well as good
psychological effects to one
Biochemical Mechanism of Depression
The causes of depression are not fully known, but it is believed that
electrochemical process in the brain are involved along with psychological
factors. Depression may be involved in an abnormal process delivering
certain important neurotransmitters.
he hormones/neurotransmitters that are associated with depression are
serotonin, norepinephrine, and dopamine. These are naturally present in
the brain and assist in communication of the nerve cells. The functions of
these neurotransmitters can be explained by the monoamine hypothesis.
This states that lowered levels of these substances can be what causes
depression.
Hormone/Neurotransmitter Imbalances leading to Depression
Depression results from monoamine neurotransmitters,
norepinephrine, serotonin and dopamine imbalance.
Serotonin is an inhibitory neurotransmitter that has been found to be
intimately involved in emotion and mood. Too little serotonin has been
BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E
shown to lead to depression, problems with anger control, obsessive-
compulsive disorder, and suicide. Too little also leads to an increased
appetite for carbohydrates (starchy foods) and trouble sleeping, which are
also associated with depression and other emotional disorders. It has also
been tied to migraines, irritable bowel syndrome, and fibromyalgia.
Norepinephrine's role as a brain and body stimulant requires a certain level
of this chemical to be present to achieve normal mental and emotional
function. Its primary mechanism is that of arousal. When low levels of
norepinephrine are present, depression symptoms may result.
Dopamine is a type of neurotransmitter created by the body and used by
the brain. It travels to various sections of the brain through separate
channels and affects several functions in the body. Dopamine is related to
hormone regulation, motor control and memory, and also increases
emotional response and sexual desire.
Theories Regarding the Cause of Depression
Monoamine Hypothesis (Advanced Monoamine Theory)
Serotonin or norepinephrine levels in the brain are regulated by MAO-A
activity mainly. However, specific symptoms of depression or mania are
related to changes in the activity of monoamine transporters in specific
brain regions. So, both MAO-A activity and density of transporters are
included in the pathophysiology of affective disorders.
Permissive Biogenic Amine Hypothesis
A deficit in central serotonergic transmission permits affective disorder, but
is insufficient for its cause; changes in central catecholaminergic
transmission, when they occur in the context of a deficit in serotonergic
transmission, act as a proximate cause for affective disorders and determine
their quality (catecholaminergic transmission being elevated in mania and
diminished in depression).
Receptor Catecholamine Hypothesis
Page 19
Supersensitivity of catecholamine receptors in the presence of low levels of D. Alcohol or Drug Intake
serotonin is the biochemical basis of depression.
May be a cause or effect of depression
Classical Norepinephrine Receptor Hypothesis
Aggravates the condition
There is increased density of postsynaptic -AR in depression.
Postreceptor Hypothesis (Neurotrophic Hypothesis (molecular and cellular
theory) of depression E. Medical Conditions

Decrease expression of brain-derived neurotrophic factor (BDNF) which is Depression is more likely to occur with certain medical conditions. These
regulated by cAMP response element binding protein (CREB) in the conditions include the following:
hippocampus. Cardiovascular disease
Causes of Depression Stroke
A. Biochemical Factors Diabetes
Chemical imbalances in the brain Cancer
Alterations in the functioning of brain chemicals called Hormonal disorders
neurotransmitters
o Thyroidism
Abnormal levels of serotonin, norepinephrine and
dopamine o Perimenopause

B. Environmental Factors Parkinsons and Alzheimers disease

academic and/or work demands

balancing school, family, work and social life

financial difficulties

peer pressure

C. Psychological Tendencies

pessimism

insecurities

self-esteem/ self-confidence issues

BIOCHEMISTRY CONFERENCE COMPILATION (FINALS) M.D. 1-E Page 20