4/28/15 5:19 AM

FORENSIC Biochemistry
The application of standard biochemical techniques and assays to
criminal cases
DNA Analysis of fingerprinting
Forensics
Means characteristic of, or suitable for a court of law
Physical Evidence
One cannot argue with physical evidence
so with DNA evidence
Person of Historical Significance
Edmond Locard (1877-1966)
o French professor
o considered the father of criminalistics
o built the worlds first forensic laboratory in France 1910
What are the Crime scene officers searching for at the scene of crime?
Evidence
LOCARDS EXHANCGE PRINCIPLE
When a suspect comes in contact with an object of person, there is
a cross-transfer of evidence
Evidence Triangle
2 Aspects of Exchange
o evidence could be left behind
o evidence could be taken away
from the crime scene
Basis of Scene of Crime Search
Locards Principle is the cornerstone of Forensic science
Locard is advanced for his time
lOCARD PRINCIPLE CAN EVEN BE APPLIED TO dna Evidence
Some examples of interchange:
o blood- drops on floor, suspect clothing, etc.
Case of Rape
Alfa Raped Bravo when bravo was unconscious
Brave did not report the crime until two weeks later- no more
evidence
Bravo became pregnant

Ngs Principle of Born Evidence

Evidence could be born
evidence could be alive
evidence could be running around
2 aspects:
o not limited to the crime scene
o the evidence can be collected even after a lapse of time
Mendel's Laws of Inheritance
Law

Definition

Dominance

Some alleles are dominant while

others are recessive, an
organism with at least one
dominant allele will display the
effect of the dominant allele

Segregation

During gamete formation, the

alleles for each gene segregate
from each other so that each
gamete carries only one allele for
each gene

Independent Assortment

Genes for different traits can

segregate independently during
the formation of gametes.

Principle of the union of nDNA

Whenever the haploid nDNA of the sperm and ovum unite in a
zygote, the totality of the united nDNA is a proof of such union.
BASIS OF PARENTAGE STUDY
United nDNA Principle
Whenever the haploid nDNA of the sperm and ovum unite in a
zygote, the totality of the united nDNA is a proof of such union
Peter Ng (2014)
Principle of Maternal mtDNA
MtDNA is inherited along the maternal line and it is an evidence of
the maternal lineage

Peter Ng (2014)
BASIS OF MATERNAL STUDY
Y chromosome is inherited along the paternal line and it is an
evidence of the paternal lineage
Maternal Analysis- mitochondrial dna
Paternal Analysis- Y chromosome
Both parents- full nuclear dna

Methods for Human Identification

DNA Profiling, or DNA fingerprinting
o Is a technique that analyzes the unique attributes of a
persons dna
o The term DNA fingerprinting was coined to allude to the
traditional use of fingerprints as a means of human
identification
o However, limited in differentiating between identical twins
(fingerprints are phenotypical)
o IDENTICAL TWINS DO NOT SHARE CLASSICAL
FINGERPRINTS
Uses of DNA Analysis
Identification of dead bodies
Evolution
Inherited diseases
Forensic cases
Paternity testing
Historial
Missing persons
Mass disasters
Military DNA Dog Tag
Convicted felon DNA databases
DNA in the Cell Nucleus
Human DNA
99% of the DNA between two individuals is identical
DNA tests look at the particular regions of the DNA that dont seem
to conserve any functionwhats commonly called junk DNA
Basic Concepts

 PCR- methods of amplifying a specific region of the genome

What type of genetic variation?
Length variation
o Short tandem repeats (STRs)
o CTAGTCGTGATAGATAGATAGCGATCGT
Sequence Variation
o Singe nucleotide polymorphisms (SNPs) insertion/deletions
o GCTAGTCGATGCTCA/T
30 LOCI REGONS
Short Tandem Repeats (STRs)
Steps in DNA Analysis
CollectionExtractionQuantification Genotyping Interpretation
of Results Database Storage and Searching
Extraction methods
Organic
o SDS, DTT, EDTA and proteinase k
Lyse the cells and extract
Chelex
o Blood stain incubate with water incubate at high
temperatures
FTA Paper
o Apply blood to paper and allow stain to dry
o Can be stored for many many years
How to separate female from the male fragment in a rape case?
Centrifuge
o Chemicals lyse the female vaginal cells, but NOT the sperm
Sperm pellet
Female supernatant
Methods
Vntr (Variable number tandem repeats )and STR
o repeated sequences of 9-18 nucleotides
STR
o repeated sequences of
Capillary Electrophoresis Method
o currently, the FBI tests 13-16 different markers
o Capillary Electropherogram

Just multiply all of the probabilities

CODIS
Combines DNA Index System
DNA Technologies
Three generation of DNA testing
Lineage Markers
o Maternal Analysis- mitochondrial dna
o Paternal Analysis- Y chromosome
o Both parents- full nuclear dna
RFLP
o you need a button-sized
PCR
o billions of copies
Mitochondrial DNA Analysis
o Mitochondrial DNA Analysis (mtDNA) can be used to examine
the DNA from samples
o Maternal lineage
since during fertilization the tail will be removed and the
mitochondria in the sperm is found in the neck, thus no
paternal mitochondrial DNA
o HIGH MUTATION RATE
DNA Analysis- Royal Family
Y Chromosomal Analysis
Mitochondrial Chromosomal Analysis
Autosomal nDNA Analysis
Sibling Study
compare the STR loci
BIOCHEMISTRY OF CONNECTIVE TISSUE
I am fed up
Connective tissue
forms the architectural framework of the body

Extracellular Matrix
Fibrous proteins
o Structural functions
keratins
collagens
elastins
o adhesive functions
fibronectin
laminin
o polysaccharide gel
Structural proteins

fibrous proteins
elongated molecules whose secondary structures are dominant
studctural motif

Collagen
from the greek word to produce glue
most abundant vertebrate connective tissue, 25%
major fibrous element
Collagen by the Diverse Forms and Functions (% weight)
Cornea 64%
liver 4%
Cartilage 50%
lung 10%
Skin 74%
Aorta 12-14%
Cortical bone 23%
Tropocollagen is a Triple Heliz
Tropocollagen
o Basic unit of the collagen fiber
o composed of 3 left-handed polypeptide chain
Collagen is rich in glycine and proline
Glycine- nearly 1/3 of all AA residues
proline about 1/4 of all AA residues
Collagend is rich in Modified aMINO aCIDS

COMPRISED NEARLY 25% OF THE TOTAL aa

PROLINE and lysine residues are covalently modified to 4hydroxyproline and 5-hydroxylysine
Hydroxylation of Proline
Needs ascorbic acid
Hydroxyproline
OH group of hYP participates in interchain h-bonding in
tropocollagen (added strength)
Formation of Hydroxylysine
Hyl seems to serve as attachment sites for polysaccharides
Ascorbic Acid

Essential to both proline hydroxylase and lysine hydroxylase

reducing agent to maintain the iron
lack of vitamin c scurvy

Scurvy
severe vit c deficiency
without hyp and hyl collagen is not assembled properly
very weak blood vessels and skin
Collagen is a Glyciprotein
Hyl residues
o Covalently attached to CHO
o Disaccharide:
Glu-Gal via alpha1,2 glycosidic bond
The Primary Structure of Collagen is Unusual (Tropocollagen)
(Gly-X-Y)
Glycine
Non polar
small
Every third residue is a glycine
Every third residue of each chain passes through the center will
only fit glyine
Proline residues are also vital to tropocollagen structure
Pyrrolidine rings repel each other by steric hindrance
Interolecular Hydrogen Bonds Stabilize Tropocollagen
Staggered peptide groups are oriented
Five Major Types of Collagen

Type

Molecular
Formula

Composition

Type I chains
(alpha1)2 alpha 2

Tissue
Distribution

remember this slide from handout

Types I,

II, III
Fibrillar collagens
main types of collagen found in connective tissues
Type I- most common
froms collagen fibrils
o thin
o cable-like
o aggregate into larger bundles or collagen fibers

Type IV
Found exclusively in the basal lamina (basement membrane)
instead of forming fibrils, it assembles into a sheet-like meshwork
Covalent Cross-links increase the mechanical strength of collagen
Tropocollagen molecules associate to form microfibrils
stabilize by both hydrogen bonds withon the triple helix and
covalent cross-links between tropocollagen tiple helics
cross-lins between lysine side side chains
Collagen Cross Links via Schiffs Base
Collagen Cross links via Aldol (?)
Covalent cross-links increase the mechanical strength of collagen
o a collagen firbre 1mm thick can hold
Stability of Collagen Helix Depends on Cooperative iNTERACTIONS
Tm- temperature at which half of the helical structure is lost
Dependence on Thermal Stability on Imino Acid Conten
The higher the imino acid content, the more stable the helix
the stability of the helix depends on the locking effect of proline and
hydroxyproline

Source

Proline plus

Ts

Tm

Body temp.

hydroxyproline
Calf Skin

232

85

65

37

Denaturation of Collagen
The Biological Assembly of Collagen Requires Many Steps
Starts in the Rough ER
o Synthesis of many chains
Hydroxylation of proline and lysine residues by vit c.
Golgi Apparatus
o chain alignment in bundles of three formation of disulfide
bonds between cysteine resides
o fORMATION OF TRIPLE-HELICLE PROCOLLAGEN
o
Formation of procollagen from 3 pro-chain
o Completion of chiains
o Packaging of procollagen into transport vesicles
o Exocytosis into the extracellular matrix
o Pro collagen will be cut by peptidase
o Formation of Tropocollagen
Removal of N- and C- terminal propeptidases (extension
peptidases)
Lateral assoc, of collagen molecules followed by
covalent cross-linking
The Collagen Biber is a Staggered Array of Tropocollagen moleculs
o Each tropocollagen molecules along a row are not linked endto-end
o holes- nucleation sites for bone 9ormation
Important Collagen Diseases
Scurvy
o Lack of Vit. C (severe Vit. C deficiency)
o without Hyp and hyl, collagen is not assembled properly
o WEAK collagen formed weak tissues

Ehler Danlos (Ehlers- Damn-loose)

o genetic disorder

o pateints have decreased of procollagen peptidase

proalpha1 will accumulate
C- and N- will not be cut, no formation of
tropocollagen
o appreciabe mounts of procollagen (pro-alpha1 and proalpha2) are present in acid extracts of skin from patients
o hypermobility of joints
o hyperflexibility of skin
o Dermatosparaxis
similar condition in cattles
Menkes Disease
o deficiency in copper results in defective crosslinking of
collagen and elastin by the copper dependent enzyme lysyl
oxidase
o Genetic disease characterized by lathyrytic symptoms due to
the inability to form the proper substrate for lysyl oxidase
Alports Syndrome
o both x-linked and autosomal genetic disorders affecting
structure of the type IV collagen fibers, the manor collagen
found in the basement membrane of the renal glomeruli
(related to laminin as discussed later)
o signs include hematuria leading eventually to end stage renal
failure
Epidermolysis bullosa
o keratin defects characterized by skin blistering
sequence abnormalities
Osteogenesis imperfecta
o Brittle bone disease
o genetic disorders characterized by bone fragility and
associated connective tisse involvement
o result form mutaion of col1 a1 and col1a2 that encode
proalpha1 and proalpha2 of type 1 collagen
o Humped-back
typical of OI

Elastin

Willams Beuren
Alpha 1 antityrpsin
Lathyrism
o disease of animals caused by ingestion of seeds of lathyris
odoratus (sweet pea)
o toxic agent- beta-aminopro

Protein with rubber-like elastic properties whose fibers can stretch

to several times their normal length without tearing
abundant in lungs, walls or large blood vessels (aorta), elastic
ligaments
small amounts in skin, tendons, loose connective tissues
irregular/random coil conformation
devoid of regular secondary structure
Distinctive amino acid composition
o 1/3 residues are glycine
o predominantyl of small, non-polar residues (alanine, valine,
leucine, isoleucine)
o > 1/3 alanine and valine, rich in proline, little
hydroxyproline
o NO HYDROXYLYSINE
Primary structure of elastin consists of alternating hydrophobic
segments (2 or 3 alanine residues and lysine-rich segments)
Lysinonorleucine
Desmosine
o Unique to elastin and responsible for its yellow color
o results from the condensation of 3 allysine and one lysine side
chain
o from condensation of 3 allysine and one lysine side chains
Elastin fibers are at least 5 times as stretchy as a rubber band of
the same cross sectional area

Collagen

Elastin

Many different genetic types

One genetic type

t

Triple helix

No triple helix, random coil

Gly-X-Y repeating structure

No repeating structure

pRESENCE of hydroxylysine

No hydroxylysine

Carbohydrat- containing

No carb

Intramolecular aldol cross links

Intamolecular desmosine crosslins

Presence of extention peptides

No extension peptides

Elastin-Associated Disorders
Willams Beurn Syndrome- deletion in the elastin gene
o rigid heart heart valve
Atherosclerosis
o Cross linking in elastin increases with age
o reduced elasticity of arterial walls
o increased incidence of obstruction to blood flow in elderly
patients
Alpha 1 antitrypsin deficiency
o Antitrypsin for Degradation for elastin
o Abraham Lincoln
rumored to have Marfan syndrome
Marfans Syndrome
o Measure arm length vs height
If arm length > height, consider Marfans syndrome
o Lens can easily be dislocated
o Michael Phelps
rumored to have Marfans syndrome
Alpha- Keratin
Mechanically durable and chemically unreactive protein
principle component in the outer epidermal layer, hair, horn, nails,
and feathers
major proteins of hair and fingernails, animal skin
form dimers- pair of alpha keratin chains
alpha keratin chains form microfibrils

o 2 dimers coiled to form a 4 molecule protofibril (basic

structure of keratin)
o 8 protofibirls microfibril (basic hair structure)
rich in cysteine- form disulfide bonds between 2 adjcent
polypeptide
PERMING
o reduction of disulfide bonds
Silk Fibroin
Beta-pleated sheet structure utilized in fibers spun by silkworms
strong and relatively inextensible
o nearly fully extended polypeptide chain

Fibrous Adhesive Proteins in Extracellular Matrix

Fibronectin
o Adhesive glycoprotein
o dimer- 2 similar subunits (2,500aa) JOINED BY A PAIR OF
DISULFIDE BONDS NEAR THEIR CARBOXYL END
o globular domains separated by regions of flexible polypeptide
chains
o 3 FORMS:
plasma fibronectin- circulates in blood, enhance blood
clotting, wound healing, and phagocytosis
cell surface fibronectin
matrix fibronectin- adhesive
o cell adhesion- contributes to the organization of the matrix
o cell migration- guide cell migration during embryogenesis
Laminin
o Like a cross
o extracellular glycoprotein
o large complex of 3 very long polypeptide chains (1500 AA)
o consists of functional domains
type IV collagen
heparin sulfate
laminin receptor proteins
o functions to anchor cells to the basal lamina enabling
epithelial cells to attach to underlying connective tissue
o Basal Lamina

BRAIN METABOLISM
Soma
o contains the nucleus
o responsible for the chemical processing of the neuron
o where neurotransmitters are produced
o Dendrites:
receive incoming information
o Axon:
carry information away from the soma to the synaptic
site (connection between neuron-neuron, muscle, or

gland)
proximal
expanded portion
axon hillock
spatial and and temporal summation
o Ramon Cajel
received Nobel Prize for research on narrow gaps
between neurons
Composition of Neuronal Membrane
o consists a large portion of lipid
o proteins
have a rapid turnover rate
o least amount is carbohydrates
o lipids have no metabolic role
for integrity of membrane
o myelin sheath
glycolipids
sphingolipids
Neurotoxins
o can interfere neuronal sodium-gated channels
o only a few would actually affect potassium-gated channels
o Cationic Guanidino group interact with Anionic Carboxylate
group located at the mouth of the Na channel on its
extracellular side
tetrodotoxin- paralytic poison from puffer fssh

Axon
o
o
o
o

saxitotoxin- from marine flagellates (red tide)

batrachotoxin- steroidal alkaloid from arrow-poison frog
specifically binds to voltage gated Na channels
renders axone membrane highly permeable to Na
can be reversed via tetrodotoxin
scorpion toxins- depolarize by binding to their Na
channels
does not compete with tetrodotoxin
textraethylammoniun- binds and blocks potassium
channels
only neurotoxin affecting K channels

Surrounded by the myelin sheath

formed from Schwann cells (PNS)
insulators
have gaps without myelin sheath (Nodes of Ranvier)
where electrical activity is confined
where salutatory conduction occurs
hastens nerve transmission
have direct contact to the surrounding ECF
o Charles Scott Sherington (?)
described the synaptic transmission
o formed from Oligoendrocytes (CNS)
Myelin Proteins
o Myelin Associated GlycoProteins
very important
part of superfamily of immunoglobulins
stabilization of the glial axon junction
lost early in multiple sclerosis
lost as MS plaques
o Po
abundant in PNS
absent in CNS
o Collexin 32
o Peripheral Myelin Protein- 22
can undergo point mutation

can cause disease Charcot Marie Tooth

progressive neuropathy
mostly the hands, legs, feet, are affected
Unmyelinated Axon
o Nerve transmission is slower vs myelinated axon
Multiple Sclerosis
o Lost of myelin sheath in the brain and spinal cord
o slows down the action potential
action potential is disseminated
there is discontinuation of transmission
o autoimmune disease or infectious

o activated T cells enter the brain and initiate the

inflammatory lesion
o intrathecal production of oligoclonal Igs
o Predisposing factors:
Disturbance of lipid and fatty acid metabolism
decreased PUFA and EFA
disturbance in FA elongation
primary increased activation of Phospholipase A1
production of lysophospholipid
one FA
disruption in muscle control, speech, and visual senses
ENERGY METABOLISM OF THE BRAIN
o Brain
2% of body mass
20% oxygen demand
60% cardiac output
requires 120 grams of glucose per day (15% energy
consumed)
primarily due to synaptic sites and astrocytes
(responsible ofr uptake of glucose)
Sodium Potassium ATPase pump
Goes against the concentration gradient
3 Na out, 2 K in
maintains membrane potential
even if brain is at rest, still active

has very insignificant Glycogen reserves

amino acids can NEVER be source of glucose
only in muscle and kidney can gluconeogenesis
occur
ketone bodies can be a source of fuel during prolonged
starvation
still needs glucose to replenish intermediates of
TCA
from acetone, acetoacetate, beta-acetylbutyrate
conversion of acetoacetate to acetyl CoA (from
succinyl CoA)

o Glutaminergic Neuron
Produces glutamate from glutamine (from astrocyte)
phosphate-activated glutaminase (PAG) is most
active here
represents a presynaptic neuron
Lactate pyruvate via NAD (pyruvate dehydrogenase)

pyruvate enters the TCA cycle

produces oxaloacetate
oxaloacetate alpha-KG

glutamate dehydrogenase is also most active here

o Astrocyte
produces alpha KG
alpha KG Glutamate
Glutamine Synthase is most active here
transferred to glutaminergic neuron
o GABA-ergic Neuron
Inhibitory neuron
GABA from Glutamate
alpha ketoglutaric acid (from TCA cycle)
glutamate decarboxylase is most active here

*glutamate is the most abundant excitatory neurotransmitter

*GDH is active in all three neurons

*Lactate from astrocyte can be transferred to Glutaminergic (presynaptic)

neuron and oxidized to pyruvate, transaminated to alanine, then back to
astrocytes
*transamination produces the glutamate (via glutamate-glutamine-lactatealanine shuttle)
Amino Acid Metabolism
GABA shunt
o Exclusive to the BRAIN
o ALPHA KG GLUTAMATE (via GDH and NADPD)
o GLUTAMATE GABA
o GABA SUCCINATE SEMI-ALDEHYDE (via transamination)
o SUCCINATE SEMIALDEHYDE SUCCINATE ( via oxidation)
*above steps BYPASS alpha KG, substrate level phosphorylation in TCA
*GABA is the most powerful inhibitory neurotransmitter

AA Shuttle
o presynaptic neuron produces glutamate
o glutamate goes to astrocytes to be converted to glutamine

Glutamate-Glutamine BCAA Shuttle

o Forms Branched-Chain AA
o Branched Chain keto acids from astrocytes to presynaptic
neuron
coupled to alpha KG- glutamate
BCAA Transferase converts BCKA to BCAA (in the
cytosol of the presynaptic neuron)
BCAA Transferase converts BCAA to BCKA (in the
mitochondrion of the astrocytes)
Resting Potential
o Depolarization
Decreasing the charge difference (-60mV to -40)
opening of sodium channels

sodium goes in
reversal of charges
opening of postassion channels
potassium moves out
o Repolarization
Increasing the charge difference
overshoot
Nernst potential
o Membrane resting potential due to ions
o delta psy- potential difference
o R- gas constant (2x10 ^ -5)

o T- absolute temperature
o F- Faradays constant (96000)
o z- charge of ion concerned
o natural log of concentration of ions outside over inside
*POTASSIUM IS THE GREATEST CONTRIBUTOR TO THE MEMBRANE
POTENTIAL
ACTION POTENTIAL
o 1. Na opens and Na ions goes in
o 2. Charge reversal
o 3. K channels opens and K goes out
o 4. Repolarization and Hyperpolarization
SYNAPSE
o 3 elements:
presynaptic neuron
releases the neurotransmitters
postsynaptic neuron
where you find the receptors
synaptic cleft
o Electrical synapse
Synaptic cleft is narrower vs chemical synapse
direct opening of the fluid channel
Action potential is sufficient in depolarization of
postsynaptic neuron
o Chemical
AP arrives at the axon of the presynaptic neuron

Neurotransmitters are released into the cleft where it

binds to the receptors in the postsynaptic neuron
ie. AcH of the myoneural junction
o Ionotropic receptors
Voltage-gated
opening and closing of the Sodium or potassium
channels
Stretch-gated
opening of pores due to the deformation of the
membrane
o Metabotropic receptors

Monomeric transmembrane protein

Has its amino and in the cleft, its carboxyl
group inside the cell
NEUROTRANSMITERS vs NEUROHORMONES
o Hormones
Long distance via the bloodstream
Effects are long-lived
Oxytocin
Vasopressin

DOPAMINE (BOTH
hormone ad
transmitter)

o Transmitters
Short-lived effect
NAME

BIOLOGICAL
EFFECTS

GnRH

Gonadotropin
Releasing Hormine

CRH

Corticotropin
releasing hormone

TRH

Thyrotropin
Releasing Hormone

HUMAN-SCALE
EFFECTS

Neurotransmitter
o Synthesized within neuron
o be released from the pre to post
o affects postsynaptic area
o exogenous sources are still considered neurotransmitter
as long as it mimics the endogenous neurotransmitters
o mechanisms exists to inactivate or remove the
transmitter from the receptor:
reuptake
degradation (enzymatic)
diffusion
Cholinergic Synapse
o AP will open voltage gated Calcium channels
o movement of presynaptic vescicles containing AcH
o release of contents of vesicles to cleft
o AcH binds to receptors in postsynaptic neuron
o Nicotinic AcH receptor- ionotropic, faster
attached to two alpha subunits
o Muscarinic AcH receptor- metabotropic
slow-acting AcH receptors
activation of G-protein (second messengers pathway)
G-alpha is directly bound to the potassium channels
efflux of potassium gives rise to long
hyperpolarization (ie. slows down the cardiac rate)
o AcH metabolism
Packed into the vesicles
Calcium (from AP) triggers the release of the vesicles
into the cleft
Acetylcholinesterase- degrades AcH for reuptake
Curare binds to the alpha subunit of the AcH receptor
Neurotoxins prevents the degradation of the AcH
promoting longer effects
o AcH Agonists

Nicotine- tobacco alkaloid, locks channels open

Decamethonium- binds nicotinic AcH receptor and locks

channels open, permanent depolarization
Succinycholine- slowly hydrozyled by AchE persistent
end plate depolarization, short-lived
Prozac- prevents reuptake of AcH
o *Anti-anxiety drugs make the receptors more sensitive (?)

o AcH Antagonist
Atropine- atropa belladonna, blocks AchAT muscarinic
receptors
D-tubocurarine- arrow poison, curare, paralytic, blocks

AcH binding site of receptor

Botulinus toxin- C, botulinum, inhibits Ach release,
Botulism
Snake Venom and anti-psychotic drugs
o Acetylcholinesterase inhibitors
Prolongs the effects of AcH
contains a serine residue
AA need
serine (200 position)
histidnie (440 position)
glutamic acid
Diisopropyl fluoriposphate (DIFP)- irreversibly
inhibits AchE by forming a stable enzyme
adduct that cannot hydrolyze AcH, permanent
depolarization
Sarin- military nerge gas, paralyzes by cholinergic
impulse bloacked, death by suffocation
Physostigmine- plant alkaloid; prevents binding of Ach
to receptor
Parathion- insecticide; electrophilic P forming a stable
enzyme product
Myasthenia Gravis
o Acquired autoimmune neuromuscular disease
o muscle weakingess due to decreased neuromuscular
transmission

o >90% found to possess antibodies to the nicotinic AchR

o drooping of eyelids at midday
o immunoglobulins prevent the AcH binding to the postsynaptic
receptors
sodium channels cannot open
AP is not elicited
Direct Neurotransmitter
o Presynaptic action potential depolarization of synaptic
terminal voltage sensitive calcium channels open calcium
enters synaptic terminal release of chemical
neurotransmitter

Indirect Neurotransmitter
o Via 2nd messengers (usually cAMP)
o usually via metabotropic

Excitatory

Inhibitory

Changes permeability of postsynaptic

neuron, stimulates membrane
depolarization

Changes iion permeability, blocks

generation of AP in postsynaptic
neuron
Opens channel proteins to admit Clincreased conductance,
hyperpolarization

Neurotransmitters Based on Chemical Structure

Amino acids

glutamate
glycine
aspartate

Biogenic amines

Gamma amiobutyrate
dopa
dopamine
norepinephrine
epinephrine
seroonine
histamine

Purine derivatives

ATP

ADP
AMP
Adenosine
Peptides

BIG GROUP
even in small quantities, can exert
great effects

CATECHOLAMINES

Glutamate Transmission
o MOST abundant excitatory neurotransmitter
o NMDA receptor
o Aminohydroxy-Methylisoxaxol-Propionic receptor
o Kinate (?) receptor
o overexcitatory is the background behind epilepsy

GABA and Glutamate Metabolism

o 3 types of receptors
GABA-A
ionotropic Cl- channels
GABA-B
metabotropic K channels
GABA-C
like GABA-A, but slower in response
o alchohol binds to GABA receptors
prolongs the quieting effect of GABA

DOPAMINE PATHWAY
o BOTH a neurotransmitter and neurohormone
o effect is in the basal ganglia (movement)
o effect is in the frontal and prefrontal area (cognition)
o effect in the nucleus accumbens and striatium (pleasure)
o COCAINE prevents the reuptake of dopamine
direct
dopamine stays longer in the synaptic cleft

 intense feeling of pleasure

can develop dependency
o OPIATES (heroine and morphine)
indirect
Binds to another neuron, decreasing GABA thus
dramatically increases the concentration of the
dopamine; goes to reward pathway leading to
intense feeling of pleasure

CATECHOLAMINE METABOLISM
o Urinary metabolite: VMA ( increased levels may indicate
adrenal medulla tumours)
SEROTONIN
o Plays a role in patients with suicidal tendencies
o 500-hyrdoxytryptamine
NEUROPEPTIDES
o Substance P- excitatory, slow pain system
o opiod peptides (endorphins)- endogenous, morphine-like
effects
o even small amounts can exert great effects
OPIODE PEPTIDES
o ALL have a YGGF tetrapeptide
o Essential for narcotic effects

COMMON NUTRITIONAL DISORDERS IN THE PHILIPPINES

Imbalance
Specific Deficiency
Vitamin A
trans configuration double bonds
generic term to describe all retinoids with biologic activity of alltrans retinol
Metabolically active:
o retinoic acid (epithelial)
o retinaldehyde (in the eye)
Vitamin Precursors

o available to the human body as carotenoid pigmentscryptoxanthines

o considered as pro0vitamin only
NO Vitamin A Activity
o Beta Carotene- MOST EFFECTIVE pro vitamin
since it will only be broken down in the small intestine
and gives rise to TWO moles of retinol
o metabolism
please refer to vit. A handouts
Retinyl esters retinol retinyl esters
beta carotene retinal retinily esters
retinyl esters chylomicron retinly esters *liver) +
retinol binding protein + transtheyretin RBP cell
surface
o funtions
medisates the visual process (retinol)
dark adaptation
supports growth and maintenance of epithelial tissues
(retinoic acid for preservation)
paraocular glands (cuboidal)
respiratory tract (pseudostratifed columnar with
goblet cells and cilia)
gastrointestinal tract (tall columnar with
microvilli)
genitourinary tract (transitional)
supporting growth and maintenance of epithelial tissues
like the skin (elascticity, over-the-counter,
TERATOGENIC)
o Deficiency
Etiologic factors
poor storage during fetal life
absence of Vit A in the diet
poor absorption of pro-vitamin A (low fat diet)
chronic gastroenteritis
malabsorption syndroimes
Early Symptoms

Nyctalopia or night blindedness

impairment of dark adaptation
due to the functional failure of the retina in
the proper regeneration of rhodopsin
more common in children
Photophopbia
eyelids are tightly closed
Xerosis conjunctivae (since the conjunctivae
epithelium is also maintained by vit A)
first clinical sign
thickening, loss of luster, frequent blinking
Bitot spots
o dry, grayish foamy plaques
o triangular or irregularly circular
o situated lateral to the cornea
o keratinized epithelial debris
REVERSIBLE
Xerophthalmia/ Corneal xerosis
cornea appears hazy or opaque with bluish
milky appearance
due to cellular infiltration
Corneal Ulcers
small erosions with opaque areola
aquaeous humor, iris and lens can escape
Keratomalacia
cornea becomes soft and gelatinous
metaplasia and degeneration of corneal
epithelium producing opacities
IRREVERSIBLE
infection
cutaneous changes
xerosis of the skin
generalized dryness with branny
desquamation
Renaderma/ Follicular Hyperkeratosis

horny papules mainly in the back of arms,

thighs, and buttocks
made of keratin
Diagnosis
thorough eye examination
biophysical examination
o dark adaptation test
biochemical tests
o plasma carotene level
< 20mcg % (low)
<10 mcg % (deficient)

o abnormal absorption test for vitamin

Prevention
RNI must be followed
Treatment
Day 1
o 110 MG RETINOL PALMITATE
o 66 mg retinol acetate orally
o 55mg retinol acetate IM injection
Day 2
Best Food Sources
butter
whole milk
fortified margarine
egg yolk
o Trace Essential Elements
Iron
adult human body contains from 3-5 grams of
iron
Dietary iron forms:
heme iron absorbed into mucosal cells as
intact porphyrin complex
non heme iron ionized by gastric juice,
reduced to ferrous and diluted with
solubilizing substances (ascorbic acid,
sugars, sulfur-containing AAs)

Mucosal transferrin
carries iron from the intestinal lumen into
the mucosal cells
combines with apoferritin inside the
mucosal cells to ferritin
Serum transferrin
carries ferric iron from mucosal cells to the
tissues
transport is affected by the size of body
stores and quantity of iron in the diet
usually saturated to about 30% (Total Iron

binding Capacity)
If iron is not needed
o transferrin is saturated
o less iron transferred from mucosal
cells
If iron is needed
o transferrin less saturated
o more iron transferred from mucosal
cells to transferrin

BIOCHEMISTRY OF VISION
Parts of the Eye
Aqueous- anterior chamber
Vitreous humour- posterior chamber
*3rd visual cell, responsible for us waking up early even without sunlight
called INHERENTLY PHOTO-SENSITIVE GANGLION CELLS
Retina pigmented epithelium
Cornea
Stroma- made up of collagen type 1 (90%) type 2 (2%) Type 6
(17%)
o Also made up of passive collagen
Fibrin-associated collagen type XII and XIV
Non- fibrillar collagens type XIII and xviii

o KERATOCYTES
o EXTRACULLAR GROUND SUBSTANCES
leucine-rich proteoglycans
decorin- dermatan SO4, PROTEOGLYCAN
Control interfibriallar spacing and the lamellar
adhesion progperties of corneal colllagens
lumican, mimecan, keratocan- involved in
regulaton of fibril diameter

Clearness and Transparency of the Cornea

Arrangement of collagen molecules
Maintenance of stromal deturgescence (Dehydrated)
soluble (NOT secreted) Vascular endothelial growth factor
receptors 3 (sVEGFR3); or else its the tyrosine-kinase
transmembrane receptors which stimulate the formation of blood
vessels in the cornea

*VASCULAR ENDOTHELIAL factors DEGFC DEFG will stimulate formation of

blood vessels, but cornea has the unique immune and angiogenic
privilege.
Collagen of Stroma
constritue more than 70% of the dry weight of the cornea
collagen fibrils are of uniform 25-35 nm diameter
o are arrainged in flash bundles called lamellae (Limbus to
Limbus)

Sodium-Potassium ATPase pumps are present (as in most cells)

*Paul boyer- BINDING chains mechanism and Gensis Ku- for Na K pump (?)

Energy source of cornea

o GLUCOSE- since eye is a special nervous tissue and nervous
tissue as we all know uses glucose primarily as energy
source
30% EMP
65% HMP
o cornea is exposed to atmospheric atmosphere, so HMP
provides NAD for the maintenance of reduced form of
glutathione reductase will react with ROS.
o METABOLICALLY ACTIVE
*lens and cornea is not provided with blood vessels, aqueous humour carries
the necessary nutrients and metabolic by-products

LENS

composition- water and proteins (crystallins)

no blood supply
metabolism- active through aquous humour
state- clear and crystalline state
unlike other

cells in the lens have NO turnover, the cells in your eye since birth will be
the same cells until your last breath.
Lens fibers

Proteins of Lens
Crystalines
o alpha (heat-shock protein; chaperones, used so that the
newly formed proteins would be folded into native
conformation; attachment of carbohydrate moieties will
determine the native conformation of the protein: Pompeiis
disease- glycogen storage disease of the alpha 1 glycosidase
enzyme)
found in many tissues with protein synthesis
mutation of which will affect not only the
*synthetic form of the myozyme was used in a Filipino, thats when they
come to know the native form of the protein via carbohydrate moieties
*LENS must be maintained in its UNAGGRETATED STATE OF THE LENS

HMP Shunt- provides NAD and EMP

Energy Source of the Lens: GLUCOSE

85% by EMP
10% by HMP
3% by TCA
*by age 40, senile cataract will occur, cataracts are the only disease of the
lens
*diabetic cataract can also cause cataracts
CATARACT
disease of the lens
opacities
cause:
o loss of normal osmolarity
o change in lens protein solubility
kinds:

o senile
o diabetic
Treatment- lens replacement
*you can opt for a bionic eye which would endow you with a vision more
than 20/20

Senile cATARACT
o cause- architectural arrangement of lens proteins altered
o breakdown of proteins molecules
starts at C-terminal
deamination
aspartyl residues racemization
o Changes include:
thiolation (cysteine), glycation, carbamylation, cysmethylation , phosphorylation and acetylation
(asparagine); also proteolysis, leading to truncation
and the release of crystalline fragments*s

DIABETIC CATARACT
*formation of sorbitol causes its accumulation in the lens
*fructose metabolism- major pathway ( in the liver) via fruktokinase and
minor pathway (in the muscle) via hexokinase

RETINA
Energy source: Glycosis
o via EMP (anaerobic)
although most of the retina is supplied by blood vessels, in the
fovea it is devoid of blood vessels, thus the need for EMP
(anaerobic) metabolism of glucose
Visual Cells:
o rods and cones (and a third visual cell: inherently
photosensitive ganglion cells)
o vascular
fovea centralis- no blood vessels
o mitochondria
outer segments of rods and cones- no mitochondria
o Enzyme
active lactate dehydrogenase

Light Conduction

*specialized cell not found in the figure: Muller cells, goes to the layer,
responsible for the deturgescence of the layers.
Rods and Cones
Visual Pigments
Rods:
o Rhodopsin
Cones:
o Red, green, or blue

outer segment- no organelles, where discs are found

inner segment- where the organelles are found
*Growth is from inner outer

*turnover happens from segment to segment, since outer segment

is destroyed by cis-retinal upon reaction with light

Transduction of Light

activation of rhodopsin metarhodopsin II

isomerization in the retinal pigmented epithelium recycles the retinal
Rhodopsin is similar to the beta-adrenergic receptors (epinephrine)

The retinal will be isomerized to all-trans which will straighten the

molecule. This will spread the helices outward

spreading of the helices via straightening of the retinal

Association of the chromophore to opsin (formation of Schiff base)

sources:

TRANSDUCIN
classical type of G protein
Trimeric: alpha, beta, gamma
Dissociation when activated, which will activate 35-cGMP
Phosphodiesterase
PHOSPHODIESTERASE (PDE6)
hetermotretrameric protein

cGMP opens the ligand-gated sodium channels (in the outer

segment), which allows entry of sodium channels
ion channels in the inner segment is not controlled by light,
responsible for transport of Na and Ca in exchange for K
when exposed to light, the cGMP levels decrease and closes the
ligand-gated sodium channels ( in the outer segment)
Na ions in the cell decreases, causing Hyperpolarization (reverse
impulsion)

Deactivation of the Phototransduction Cascade

GTPase Activating protein (GAP) S-subunit
Gunaylate Cyclase Activating protein (GCAP)
Deactivation of Metarhodopsin II
o recoverin and arrestin

CONES
color vision- trichromatic
Three types of cells defines by visiual pigments;
o cyanopsin- blue- 420nm
o iodopsin- green- 535 nm
o porphyrhodopsin- red- 565 nm

Color Vision is Trichromatic:

Deuteronopria- absence of iodopsin (green) (medium)
Protonopia- absence of prophyropsin (red) (long)
tritanopia- absence of cyanopsin (blue) (short)
*John Dalton was` color-blind (deuteronopia), his last will and testament
was to pluck his eyes out and to find what was wrong with it. It was kept in
a museum for many years. A scientist used recombinant dna to discover that
john Dalton was color-blind
Genes of Visual Pigments:
o Rhodopsin- Chromosome 3
o Blue pigment- Chromosome 7
o Red and Green pigment- Chromosome X

4/28/15 5:19 AM
ISOLATION, EXTRACTION, CHARACTERIZATION AND
AMPLIFICATION OF DNA
human Genomic DNA
23 pairs of chromosomes
3 billion base pairs
~30-50k GENES
~5% codings (exons) and 95% non coding (introns)
Exons are expressed, introns are not
Isolation and extraction of geniomic DNA
o from cheek cells and hair follicles
Amplification of DNA by PCR
Agarose gel electrophoresis of amplified PCR samples and staining
Analysis
Isolation and Extraction of DNA
All cells contiain identical set of DNA
Basic procedure include isolation and lysi of cells, precipitation and
extraction of DNA
InstaGene matrix chelate ions (cofactors of enzymes which may
degrade the DNA)
Extraction
Cheek cell DNA
o Saline mouthwash collection
Hair follicle DNA
o Hair trimmed 2cm from bublb protease
Pre-incubation at 56 degrees Celsius, 10 min
o Know the significance of the temperature
Incubation at 100 degrees Celsius, 5 min
o Know the significance of the temperature
Centrifugation at 6000X g, 5 min
Supernatant collection
Quantification of Exracted DNA
Optical density readin at 260
1 OD reading = 50 ug/ml
dilution factor (100)
OD reading at 280 (DNA) and 260 (protein) (?)

GET THE RATIO TO DETERMINE THE PURITY (1.5, more dna than
protein)

Cheek cell /hair DNA template

Master mix:
o dNTPs, Taq polymerase, buffer (Mg++ as cofactor)
Forward and reverse primer (tartrazine dye-yellow): 50~bp
Thermocycler:
o 94 degrees Celsius (denaturation)
o 60 degrees Celsius (annealing)
o 72 degrees Celsius (extension)

PCR

Target Sequence: Alu

Stands for restriction enzyme Alu
o Small, repetitive DNA elements of ~300 bp repeated almost
500,000x (SINEs), an INTRON
o Randomly inserted over million of years
o simply a measure of molecular genetic variation
o PV92 region of chromosome 6
Target Sequence
641 base pairs- absence of Alu segment
941 base pairs- presence of Alu segment
Agarose Gel Electrophoresis
Separates DNA fragments based on size
Agarose 1%
1XTAE running buffer covers entire gel (Tris buffer)
Loading dye included in samples monitor migration of DNA
(samples and standards)
Fragments move toward anode
Gel stained with Fast Blast
PCR of PV92 Region of Chromosome 16
Alu is dimorphic
Alu sequence inserted within intron (641 bp)
o Geetic variations
Alu insert within PV92 region of Chromosome 16
PV92

Genotype

DNA SIZE and PCR

Products
Homozygous (+/+)
Heterozygous (+/-)
Homozygous (-/-)
Empty Lanes or Unamplified sAMES

941 pb

4/28/15 5:19 AM