Biochemistry

Guide Questions:
1. Discuss the fundamental unit of muscle and its myofibril.
2. What are the major proteins of muscle and their form?
3. What are the major biochemical events occurring during a cycle of a muscle contraction and relaxation?
4. Discuss the synthesis, uptake and degradation of acetylcholine in the neuromuscular junction.
5. Describe the structure of the botulism toxin, and how it is activated.
6. How does botulism toxin cause food poisoning? Discuss its main role in the pathophysiology of the symptoms show in the
case.
Answers:
1. Muscles:
Each muscle is composed of numerous cells called muscle fibers.
A connective tissue layer called the endomysium surrounds each of these fibers.
Individual muscle fibers are then grouped together into fascicles, which are surrounded by another connective tissue
layer called the perimysium.
Finally, fascicles are joined together to form the muscle. The sheath that surrounds the muscle is called the
epimysium.
Each skeletal muscle fiber contains bundle of filaments, called myofibrils.
Myofibrils are organic cable-like structures composed of essential proteins. Within the myofibrils are bundles of
these proteins, arranged into thick and thin filaments within repeating sections known as sarcomeres.
2. Major proteins of muscle:
= Myomesin
A myosin-binding protein which holds the thick filament in place
= F- actin
Compose the thin filaments of the muscles
Consists of long filamentous polymers containing two strands of globular (G-actin) monomers, twisted
around each other in a double helical formation.
= G-actin
Asymmetric and polymerize to produce a filament with polarity
Each monomer contains a binding site for myosin
= Tropomyosin
A long, thin molecule containing 2 polypeptide chains, which assembles to form a long polymer located in
the groove between the 2 twisted actin strands.
= Troponin
It is a complex of 3 subunits:
- TnT attaches to tropomyosin
- TnC binds calcium ions
- TnI inhibits the actin-myosin interaction
= Myosin
It can be dissociated into 2 identical heavy chains and 2 pairs of light chains.
Myosin heavy chains are thin, rod-like molecules made up of 2 heavy chains twisted together as myosin
tails.
Small globular projections at one end of each heavy chain forms the heads, which have ATP binding sites.
The 4 light chains are associated with the head.
= Titin
Largest known muscle protein
A single chain of 27000 amino acids with a molecular weight of about 3000kDa
= Nebulin
A muscle protein closely associated with the actin filaments
May have a role in determining their length
3. Muscle contraction and relaxation:
o The motor neuron initiates contraction of skeletal muscle by producing an action potential in the muscle fiber.
o As the action potential passes down the T tubules of the muscle fiber, dihydropyridine receptors (DHPRs) in the T
tubules undergo conformational changes that result in the opening of neighboring SR Ca
++
channels called
ryanodine receptors (RYRs), which then release Ca
++
to the myoplasmic Ca
++
from the SR.
o The increase in myoplasmic Ca
++
promotes muscle contraction by exposing myosin binding sites on the actin thin
filaments.
o Myosin cross-bridges then appear to undergo a ratchet action, with the thin filaments pulled toward the center of the
sarcomere and contracting the skeletal muscle fiber.
o Relaxation of the muscle follows as myoplasmic Ca
++
is resequestered by Ca
++
-ATPase (SERCA) in the SR. Uptake
of Ca
++
into the id due to the action of a Ca
++
pump.



4. Acetylcholine:
+ It is synthesized from acetyl coenzyme A and choline by the enzyme choline acetyltransferase, which is located in
the cytoplasm of cholinergic presynaptic terminals.
+ After synthesis, acetylcholine is concentrated in vesicles.
+ After release, the action of acetylcholine is terminated by the enzyme acetylcholinesterase, which is highly
concentrated in the synaptic cleft.
+ Acetylcholinesterase hydrolyzes acetylcholine into acetate and choline.
+ The choline is then taken up by a Na
+
symporter in the presynaptic membrane for the resynthesis of acetylcholine.
+ The extracellular degradation of acetylcholine is unusual for a neurotransmitter inasmuch as the synaptic action of
other classic neurotransmitters is terminated via reuptake by a series of specialized transporter proteins.
5. Structure of botulism toxin:
Botulinum toxins are colorless, odorless, and presumably tasteless.
The toxins are produced by vegetative cells (i.e., germination of spores) and released by cell lysis.
The botulinum toxin molecule is synthesized as a single chain and then cleaved to form the dichain molecule with a
disulfide bridge.
The light chain acts as a zinc endopeptidase similar to tetanus toxin with proteolytic activity located at the N-
terminal end. The heavy chain provides cholinergic specificity and is responsible for binding the toxin to presynaptic
receptors; it also promotes light-chain translocation across the endosomal membrane.
Some toxins are fully activated by the bacteria that produce them (proteolytic strains of type A, B, and F), and some
require exogenous proteolytic activation (types E and non-proteolytic types B and F).
6. The clinical syndrome of botulism can occur following ingestion of contaminated food. Following exposure, pathogenesis
includes the following steps:
O Botulinum toxin is activated by proteolytic cleavage; the activated structure is a 150-kd polypeptide comprising two
chains (a heavy chain and a light chain) that are connected by a single disulfide bond.
O Botulinum toxin enters the circulation and is transported to the neuromuscular junction.
O At the neuromuscular junction, the heavy chain of the toxin binds to the neuronal membrane on the presynaptic side
of the peripheral synapse.
O The toxin then enters the neuronal cell via receptor-mediated endocytosis.
O The light chain of the toxin crosses the membrane of the endocytic vesicle and enters the cytoplasm.
O Once inside the cytoplasm, the light chain of the toxin (which is a zinc-containing endopeptidase) cleaves some of
the proteins that form the synaptic fusion complex. The synaptic proteins, referred to as SNARE proteins, include
synaptobrevin (cleaved by toxin types B, D, F, and G), syntaxin (cleaved by toxin type C), and synaptosomal-
associated protein (SNAP-25; cleaved by toxin types A, C, E). The clostridial neurotoxin apparently first binds to
the SNARE complex before cleavage occurs.
O The synaptic fusion complex allows the synaptic vesicles (which contain acetylcholine) to fuse with the terminal
membrane of the neuron. Disruption of the synaptic fusion complex prevents the vesicles from fusing with the
membrane, which in turn prevents release of acetylcholine into the synaptic cleft.
O Without neuronal acetylcholine release, the affiliated muscle is unable to contract and becomes paralyzed.
O The blockade of acetylcholine release lasts up to several months; normal functioning slowly resumes either through
turnover of SNARE proteins within the cytoplasm or through production of new synapses.
O Death from botulism results acutely from airway obstruction or paralysis of respiratory muscles. Death also can
result from complications related to prolonged ventilator support and intensive care.
O Botulinum toxin apparently does not cross the blood-brain barrier; therefore, central nervous system functions
remain intact.
Microscopic Human Structural Biology
Guide Questions:
1. What are the different types of muscle?
2. Enumerate and describe the different parts of these muscle types.
3. Illustrate and label the parts of a neuron.
4. What is a neuromuscular junction?
a. What is the effect of botulism toxin in the NMJ?
5. Enumerate and describe the parts of a neuromuscular junction.
6. What are the common neurotransmitters?
7. Differentiate Somatic from Autonomic Nervous System.
8. Describe and differentiate the types of the autonomic nervous system.
Answers:
1. There are three types of muscle tissue:
a. Skeletal muscle
Anchored by tendons (or by aponeuroses at a few places) to bone and is used to effect skeletal movement
such as locomotion and in maintaining posture.
b. Smooth muscle
Found within the walls of organs and structures such as the esophagus, stomach, intestines, bronchi, uterus,
urethra and bladder
Unlike skeletal muscle, smooth muscle is not under conscious control.
c. Cardiac muscle
Also an "involuntary muscle" but is more akin in structure to skeletal muscle
Found only in the heart
2. Muscle types and their different parts:
+ Each muscle is composed of numerous cells called muscle fibers.
+ A connective tissue layer called the endomysium surrounds each of these fibers.
+ Individual muscle fibers are then grouped together into fascicles, which are surrounded by another connective tissue
layer called the perimysium.
+ Finally, fascicles are joined together to form the muscle. The sheath that surrounds the muscle is called the
epimysium.
+ Each skeletal muscle fiber contains bundle of filaments, called myofibrils.
+ Myofibrils are organic cable-like structures composed of essential proteins. Within the myofibrils are bundles of
these proteins, arranged into thick and thin filaments within repeating sections known as sarcomeres.
+ The sarcomere is demarcated by 2 dark lines called Z lines and represents a repeating contractile unit in skeletal
muscles.
+ On either side of a Z line is a light band (I band) that contains thin filaments composed primarily of actin.
+ The area between two I bands within a sarcomere is the A band, which contain thick filament composed primarily of
myosin.
+ A light area present in the center of the sarcomere is called the H band.
+ A dark line called M line is evident in the center of the sarcomere.
3. Parts of a Neuron
4. Neuromuscular junction is a specific type of synapse that occurs between motor neuron and muscle fiber.
Botulinum toxin enters the circulation and is transported to the neuromuscular junction.
At the neuromuscular junction, the heavy chain of the toxin binds to the neuronal membrane on the presynaptic side of
the peripheral synapse.
The toxin then enters the neuronal cell via receptor-mediated endocytosis.
The light chain of the toxin crosses the membrane of the endocytic vesicle and enters the cytoplasm.
Once inside the cytoplasm, the light chain of the toxin (which is a zinc-containing endopeptidase) cleaves some of the
proteins that form the synaptic fusion complex. The synaptic proteins, referred to as SNARE proteins, include
synaptobrevin (cleaved by toxin types B, D, F, and G), syntaxin (cleaved by toxin type C), and synaptosomal-associated
protein (SNAP-25; cleaved by toxin types A, C, E). The clostridial neurotoxin apparently first binds to the SNARE
complex before cleavage occurs.
The synaptic fusion complex allows the synaptic vesicles (which contain acetylcholine) to fuse with the terminal
membrane of the neuron. Disruption of the synaptic fusion complex prevents the vesicles from fusing with the
membrane, which in turn prevents release of acetylcholine into the synaptic cleft.
Without neuronal acetylcholine release, the affiliated muscle is unable to contract and becomes paralyzed.
5. Parts of a Neuromuscular junction:
a. Synaptic end bulbs:
At the end of each axon terminal, there is a bulbous swelling called synaptic end bulb.
Each synaptic end bulb contains many synaptic vesicles.
These vesicles contain the all-important neurotransmitter substances such as acetylcholine. These
neurotransmitter substances are responsible for transmission of impulse from axon to muscle fiber through
the synapse.
b. Motor end plate:
It is the part of the sarcolemma of muscle cell, which is in closest proximity to the synaptic end bulb.
It shows certain specific features different than those of other regions of muscle cell sarcolemma,
including:
Synaptic Gutter: It is the invaginated membrane, which forms space for the synaptic end bulbs to reach
close to the muscle fiber sarcolemma.
Subneural Clefts: These are small folds of the muscle membrane present at the bottom of the synaptic
gutter. They greatly increase the surface area at which the neurotransmitter can act.
Increased number of mitochondria: The area of the muscle fiber surrounding the motor end plate shows
a considerable increase in the number of mitochondria. The obvious reason for this is the energy demand of
the neuromuscular junction.
c. The Synaptic cleft:
It is the space between the motor end plate (muscle fiber part) and synaptic end bulb (motor neuron part) of
the neuromuscular junction.
Because of this cleft, the connection between the motor neuron and the muscle fiber is not continuous and
there is a break. This break is traversed by the neurotransmitters.

6. Common neurotransmitters:
a. Glutamate
o It is used at the great majority of fast excitatory synapses in the brain and spinal cord.
o Excessive glutamate release can lead to excitotoxicity causing cell death.
b. GABA
o It is used at the great majority of fast inhibitory synapses in virtually every part of the brain.
o Many sedative/tranquilizing drugs act by enhancing the effects of GABA.
c. Acetylcholine
o It is distinguished as the transmitter at the neuromuscular junction connecting motor nerves to muscles.
o Acetylcholine also operates in many regions of the brain, but using different types of receptors.
d. Dopamine
o It has a number of important functions in the brain.
o It plays a critical role in the reward system, but dysfunction of the dopamine system is also implicated in
Parkinson's disease and schizophrenia.
e. Serotonin
o It is a monoamine neurotransmitter.
o It functions to regulate appetite, sleep, memory and learning, temperature, mood, behaviour, muscle
contraction, and function of the cardiovascular system and endocrine system.
o It is speculated to have a role in depression.
f. Substance P
o It is an undecapeptide responsible for transmission of pain from certain sensory neurons to the central
nervous system.
7. Somatic Nervous System and Autonomic Nervous System
a. Somatic nervous system
It is the part of the peripheral nervous system that is responsible for carrying motor and sensory information
both to and from the central nervous system.
This system is made up of nerves that connect to the skin, sensory organs and all skeletal muscles.
The system is responsible for nearly all voluntary muscle movements as well as for processing sensory
information that arrives via external stimuli including hearing, touch and sight.
b. Autonomic Nervous System
It is the part of the peripheral nervous system that acts as a control system functioning largely below the
level of consciousness, and controls visceral functions.
The ANS affects heart rate, digestion, respiratory rate, salivation, perspiration, pupillary dilation,
micturition (urination), and sexual arousal.
Whereas most of its actions are involuntary, some, such as breathing, work in tandem with the conscious
mind.
8. Types of Autonomic Nervous System:
a. Sympathetic nervous system
Promotes a "fight or flight" response, corresponds with arousal and energy generation, and inhibits
digestion.
- Diverts blood flow away from the gastro-intestinal (GI) tract and skin via vasoconstriction.
- Blood flow to skeletal muscles and the lungs is enhanced.
- Dilates bronchioles of the lung, which allows for greater alveolar oxygen exchange.
- Increases heart rate and the contractility of cardiac cells (myocytes), thereby providing a
mechanism for the enhanced blood flow to skeletal muscles.
- Dilates pupils and relaxes the ciliary muscle to the lens, allowing more light to enter the eye and
far vision.
- Provides vasodilation for the coronary vessels of the heart.
- Constricts all the intestinal sphincters and the urinary sphincter.
- Inhibits peristalsis.
b. Parasympathetic nervous system
Promotes a "rest and digest" response, promotes calming of the nerves return to regular function, and
enhances digestion.
- Dilates blood vessels leading to the GI tract, increasing blood flow. This is important following
the consumption of food, due to the greater metabolic demands placed on the body by the gut.
- The parasympathetic nervous system can also constrict the bronchiolar diameter when the need for
oxygen has diminished.
- Dedicated cardiac branches of the Vagus and thoracic Spinal Accessory nerves impart
Parasympathetic control of the Heart or Myocardium.
- During accommodation, the parasympathetic nervous system causes constriction of the pupil and
contraction of the ciliary muscle to the lens, allowing for closer vision.
- The parasympathetic nervous system stimulates salivary gland secretion, and accelerates
peristalsis, so, in keeping with the rest and digest functions, appropriate PNS activity mediates
digestion of food and indirectly, the absorption of nutrients.

Gross Human Structural Biology
Guide Questions:
1. Discuss briefly the cause and pathophysiology of the disease.
2. Discuss briefly the mode of transmission.
3. For the following common signs/symptoms of Botulism, specify the involved organs/structure and their gross features and
functions.
a. Medial Strabismus
b. Mydriasis
c. Nystagmus
d. Diplopia with blurred vision
e. Ptosis
f. Dysphagia
g. Diminished gag reflex
h. Dysarthria
i. Dysphonia
j. Descending Flaccid Paralysis
k. Incoordination
l. Irregular Respiration
m. Generalized Weakness
4. Discuss prevention, treatment and prognosis of disease.
Answers:
1. Exposure to botulinum toxin occurs through the following mechanisms (toxin is not absorbed through intact skin):
a. Ingestion of pre-formed toxin
b. Inhalation of pre-formed toxin
c. Local production of toxin by C botulinum organisms in the gastrointestinal tract
d. Local production of toxin by C botulinum organisms in devitalized tissue at the site of a wound
e. Iatrogenic exposure caused by injection of botulinum toxin for cosmetic purposes or to treat certain musculoskeletal
disorders, such as spasticity or blepharospasm
Following exposure, pathogenesis includes the following:
Botulinum toxin is activated by proteolytic cleavage; the activated structure is a 150-kd polypeptide comprising two
chains (a heavy chain and a light chain) that are connected by a single disulfide bond.
Botulinum toxin enters the circulation and is transported to the neuromuscular junction.
At the neuromuscular junction, the heavy chain of the toxin binds to the neuronal membrane on the presynaptic side
of the peripheral synapse.
The toxin then enters the neuronal cell via receptor-mediated endocytosis.
The light chain of the toxin crosses the membrane of the endocytic vesicle and enters the cytoplasm.
Once inside the cytoplasm, the light chain of the toxin (which is a zinc-containing endopeptidase) cleaves some of
the proteins that form the synaptic fusion complex. The synaptic proteins, referred to as SNARE proteins, include
synaptobrevin (cleaved by toxin types B, D, F, and G), syntaxin (cleaved by toxin type C), and synaptosomal-
associated protein (SNAP-25; cleaved by toxin types A, C, E). The clostridial neurotoxin apparently first binds to
the SNARE complex before cleavage occurs.
The synaptic fusion complex allows the synaptic vesicles (which contain acetylcholine) to fuse with the terminal
membrane of the neuron. Disruption of the synaptic fusion complex prevents the vesicles from fusing with the
membrane, which in turn prevents release of acetylcholine into the synaptic cleft.
Without neuronal acetylcholine release, the affiliated muscle is unable to contract and becomes paralyzed.
The blockade of acetylcholine release lasts up to several months; normal functioning slowly resumes either through
turnover of SNARE proteins within the cytoplasm or through production of new synapses.
2. Botulism transmission varies based on the type of botulism. Unlike infectious diseases, however, botulism transmission does
not occur from one person to another.
a. Foodborne Botulism Transmission
. Foodborne botulism transmission occurs through eating foods contaminated with botulinum spores that grow
into bacteria and produce botulism toxins in the food.
. A common cause of botulism food poisoning is improperly preserved home-processed foods with low acid
content.
b. Wound Botulism Transmission
. Wound botulism transmission occurs when bacteria that cause botulism (Clostridium botulinum) contaminate a
wound, germinate, grow within the wound, and produce toxin that is absorbed into the bloodstream.
c. Infant Botulism Transmission
. Infant botulism transmission differs from foodborne botulism in that the toxins themselves are not ingested.
. Instead, Clostridium botulinum spores are swallowed by the infant.
. Spores allow bacteria to survive in a dormant state until exposed to conditions that can support their growth.
. When consumed, these spores turn into bacteria in the favorable environment of the baby's large intestine and
produce a toxin responsible for symptoms of the disease.

3. Common Signs and Symptoms of Botulism:
a. Medial Strabismus - inability of the eyes to focus together because of an imbalance in the muscles that control eye
movement
b. Mydriasis - dilation of the pupil
c. Nystagmus - is a term to describe fast, uncontrollable movements of the eyes
d. Diplopia with blurred vision - commonly known as double vision, is the simultaneous perception of two images of a
single object that may be displaced horizontally, vertically, or diagonally in relation to each other.
e. Ptosis - is a drooping or falling of the upper or lower eyelid
Organ/structure involved: Eyes
As a conscious sense organ, the eye allows vision.
The eye is not properly a sphere, rather it is a fused two-piece unit. The smaller frontal unit, more curved,
called the cornea is linked to the larger unit called the sclera. The corneal segment is typically about 8 mm
(0.3 in) in radius. The sclerotic chamber constitutes the remaining five-sixths; its radius is typically about
12 mm. The cornea and sclera are connected by a ring called the limbus. The iris the color of the eye
and its black center, the pupil, are seen instead of the cornea due to the cornea's transparency.
f. Dysphagia - difficulty in swallowing
g. Diminished gag reflex
h. Dysarthria - s a condition in which you have difficulty controlling or coordinating the muscles you use when you
speak, or weakness of those muscles
i. Dysphonia - an impairment in the ability to produce voice sounds using the vocal organs
Organ/structure involved: Larynx, Pharynx
The laryngeal skeleton consists of nine cartilages: three single (epiglottic, thyroid and cricoid) and
three paired (arytenoid, corniculate, and cuneiform).
The larynx, commonly called the voice box, is an organ in the neck which is involved in
breathing, sound production, and protecting the trachea against food aspiration.
It manipulates pitch and volume. The larynx houses the vocal cords, which are essential for
phonation.
The pharynx is a fibromuscular tube which extends from the base of the skull to the lower border
of the cricoid cartilage (at which point it becomes the esophagus).
Portions of the pharynx lie posterior to the nasal cavity (nasal pharynx), oral cavity (oral pharynx)
and larynx (laryngeal pharynx). The muscular walls of the pharynx are comprised of an outer layer
made up of 3 circularly disposed muscles, the constrictors.
During swallowing, successive contraction of the superior, middle, and inferior constrictor
muscles helps to propel the bolus (ball) of food down into the esophagus. In addition, contraction
of the 3 longitudinal muscles of the pharynx helps to raise the pharynx, effectively aiding it in
engulfing the bolus of food.
In between acts of swallowing, the lowest fibers of the inferior constrictor are thought to act as a
sphincter, guarding the entrance to the esophagus and preventing the entry of air into the digestive
system.
j. Descending Flaccid Paralysis
k. Incoordination
Organ/structure involved: Skeletal muscles
Muscle fibers are connected to the skeletal system by tendons and other tissues.
Skeletal muscle is made up of individual components known as myocytes.
Their function is to produce force and cause motion.
l. Irregular Respiration
Organ/structure involved: Lungs
The lungs are located in two cavities on either side of the heart.
Though similar in appearance, the two are not identical. Both are separated into lobes by fissures,
with three lobes on the right and two on the left.
Their principal function is to transport oxygen from the atmosphere into the bloodstream, and to
release carbon dioxide from the bloodstream into the atmosphere.
m. Generalized Weakness
Organ/structure involved: Skeletal muscles
Muscle fibers are connected to the skeletal system by tendons and other tissues.
Skeletal muscle is made up of individual components known as myocytes.
Their function is to produce force and cause motion.
4. Prevention, Treatment and Prognosis of Botulism:
a. Prevention
- Use proper canning techniques
D Be sure to use proper techniques when canning foods at home to ensure that any botulism germs in
the food are destroyed:
D Pressure cook these foods at 250 F (121 C) for at least 30 minutes.
D Consider boiling these foods for 10 minutes before serving them.
- Prepare and store food safely
D Don't eat preserved food if its container is bulging or if the food smells spoiled. However, taste
and smell won't always give away the presence of C. botulinum. Some strains don't make food
smell bad or taste unusual.
D If you wrap potatoes in foil before baking them, eat them hot or store them in the refrigerator
not at room temperature.
D Store oils infused with garlic or herbs in the refrigerator.
- To reduce the risk of infant botulism, avoid giving honey even a tiny taste to babies under the age of
1 year.
- To prevent wound botulism and other serious bloodborne diseases, never inject or inhale street drugs.
b. Treatment
- Supportive care is the mainstay for treatment of botulism; prolonged intensive care, mechanical ventilation,
and parenteral nutrition may be required.
- Botulinum antitoxin can be administered to treat forms of botulism other than infant botulism and is most
effective if given early in the clinical course. Although antitoxin will not reverse existing paralysis, it will
prevent additional nerve damage if given before all circulating toxin is bound at the neuromuscular
junction.
- In cases of wound botulism, the wound should be surgically debrided and antibiotics should be
administered (usually penicillin).
c. Prognosis
- Recovery from botulism is often prolonged and may require extensive rehabilitation.
- Ventilatory muscle recovery invariably occurs but it is slow and variable. Although ventilatory muscle
strength is usually normal by 1 year, exercise capacity is often reduced.
- Patients may complain of marked fatigue, general weakness, dry mouth and shortness of breath that may
persist for over a year, particularly if infected with type B botulinum toxin.
- Death from botulism results acutely from airway obstruction or paralysis of respiratory muscles.
- Death also can result from complications related to prolonged ventilatory support and intensive care.
- Botulinum toxin apparently does not cross the blood-brain barrier; therefore, central nervous system
functions remain intact.