Sabotage of The Cell Signal

Sabotage of the Cell Signal Curriculum
Curriculum Introduction

Lesson 1
Teacher Instructions
Group Assignments
Scenario Pt. 1
Scenario Pt. 2
Scenario Pt. 3 Gment Response
Scenario Pt. 3 Hospital Personnel
Scenario Pt. 3 Labs
Scenario 3 Questions

Lesson 2
ELISA Plates
Student Instructions
Data Sheet

Lesson 3
Science Lab Report Rubric
Student Instructions

NIH Curriculum: Sabotage of the Cell Signal - A teaching module on the effect of
Anthrax toxins on cell signaling.

Introduction

The signaling systems discussed in this module are only a few of the many ways that cells send
messages between cells and the extracellular matrix, and within themselves. Many of the
signaling pathways are interconnected with one another, creating an incredibly complex
system. Though the system is complex, there are two main points that bear describing. One of
the most important aspects of cell signaling regards the protein macromolecules that make are
involved in all three stages of signaling, reception, transduction, and response. The second
major issue in conducting a message through or between cells involves the many steps that is
needed to pass molecule to molecule and create the important cascade that is needed. Each of
the following activities in this module fit together to drive home the complexity of the system,
and how health and community can be affected if the system is interrupted.

Background Information

Cell Signaling
Cell to cell communication is essential for multicellular organisms because they
must coordinate their activities. Cells most often communicate with each other
by chemical signals. Communicating cells may be close together or far apart.
Cells in a multicellular organism usually communicate by releasing chemical
messengers targeted for cells that may not be immediately adjacent. Local
regulators are chemicals that stimulate cells nearby, such as the release of growth factors or
axon terminals releasing neurotransmitters into a synapse to stimulate receptor cells. Cells can
also communicate through direct contact where cell membrane molecules connect, such as
developing cells in an embryo or immune system cells. Specialized proteins called integrins are
present in the plasma membrane to mediate attachment to the extracellular matrix or
neighboring cells.

The three stages of cell signaling are reception, transduction, and response. A signal molecule
binds to a receptor protein, causing the protein to change shape. Only certain, specific, target
cells can detect and react to a signal due to receptors complementary to the signal molecule.
Signal molecules work like a ligand, any small molecule that binds to a specific larger molecule,
and often stimulate the receptor molecule to change shape.

Often this new shape fits the shape of other molecules within a cell. When they bind, the
internal molecule now becomes active. Most signal receptors are plasma membrane proteins
that are water-soluble and too large to pass freely through the plasma membrane so they must
bind to receptors on the outside of a cell. This receptor then transmits information into the
cell. There are three major types of cell membrane receptors.

a) G-Protein-Linked Receptor is a plasma membrane receptor that activates G proteins within
the cytoplasm by removing GDP and replacing it with GTP. The G protein is now active and
can bind to another protein, usually an enzyme, in order to stimulate the next step in the
pathway.

This lasts only a short while because the activated G protein also functions as an enzyme
that removes its GTP molecule and replaces it with GDP, to return itself back to the inactive
state. Without the GTP, the G protein will also separate from the enzyme. G protein
receptor systems function during embryonic development, vision and smell. Some bacteria
toxins cause disease by blocking G proteins, and therefore blocking the next step in a signal
pathway.

b) Tyrosine-Kinase Receptors have enzymatic activity within a cell. For example, when a
growth factor ligand binds to tyrosine-kinase receptors on the outside of a plasma
membrane it stimulates two separate polypeptides to bind into one active molecule.
The part extending into the cytoplasm now functions as an active enzyme to add
phosphates to its tyrosine amino acids. This activates the receptor protein so it can activate
relay proteins within the cytoplasm. One of these activated tyrosine-kinase dimers has the
ability to activate ten different relay proteins that can activate different signal-transduction
pathways.
c) Ion-Channel Receptors are channel proteins in a plasma membrane that can open or close
in response to a signal from a ligand. These channels are important in the functioning of the
nervous system.
d) Intracellular Receptors are proteins dissolved in the cytosol or nucleus of target cells.
Because they are in the cytoplasm their signal ligand must be able to pass through the
plasma membrane. Steroids and nitric oxide (NO) have this ability. For example, the male
hormone testosterone (a steroid) is released by the testes, passes into a target cell, and
binds with a receptor protein in the cytoplasm to activate it. This active protein is now a
transcription factor that can bind to a promoter and initiate transcription of mRNA.

Pathways relay signals from receptors to cellular responses
1. Proteins are the most common relay molecules in signal transduction pathways. Stimulus by
one signal ligand can be amplified many times as it is passed along a pathway.
Often these relay proteins are activated by phosphorylation. Protein phosphorylation, a
common mode of regulation in cells, is a major mechanism of signal transduction
The general name for an enzyme that transfers phosphate groups is protein kinase. They
differ from tyrosine kinase proteins in that they phosphorylate other proteins, while
tyrosine-kinase phosphorylates itself. Many of the relay molecules in signal transduction
pathways are protein kinases that work by phosphorylating each other in a cascading
sequence. Enzymes called protein phosphatases function to remove phosphates from
activated protein kinases, thus turning off a pathway.

2. Certain small molecules and ions are key components of signaling pathways. Many
signaling pathways involve small, non-protein, water-soluble molecules or ions called
second messengers. Once a ligand has attached to a plasma membrane receptor and
activated it, a second messenger such as cyclic AMP or calcium ions (Ca2+) can function in a
pathway by moving rapidly throughout the cytosol.
Cyclic AMP is a modified ATP. For example, epinephrine stimulates a G protein receptor that
activates a G protein that activates an enzyme embedded in the plasma membrane. This
activated enzyme converts ATP into cyclic AMP (cAMP) which in turn activates a protein
kinase. The activated protein kinase can now phosphorylate other proteins in its pathway in
order to lead to glycogen conversion into glucose in the liver and provide fuel for fight or
flight. Another enzyme in the cytoplasm functions to inactivate cAMP and keep the
signaling pathway from going on indefinitely.

Inactivates
Epinephrine cAMP
G protein receptor
Activates G protein
Activates enzyme in membrane
Converts ATP to cAMP
Activates protein kinase
Phosphorylates
other proteins

An example of the effects caused by interrupting the signaling pathway can be seen by the
toxins released by the Vibrio cholera bacteria that keep G proteins active. These active G
proteins keep stimulating the production of cAMP and stimulate the cells lining our intestines
to secrete large amounts of water and salts. This can lead to diarrhea, dehydration and death,
especially in infants.

Calcium functions as a second messenger more often than cAMP. Increasing the concentration
of Ca2+ in the cytoplasm can stimulate muscle cell contraction, cell division and secretion of
other substances. Ca2+ functions as a second messenger in both G protein and tyrosine-kinase
pathways. Animals have high concentrations of Ca2+ in their interstitial fluid and within their
endoplasmic reticulum. There are low concentrations in the cytosol. Slight changes in cytosol
concentration can activate a protein in a signal transduction pathway.
In some cases, an activated G protein can stimulate the production of a second messenger
called Inositol Triphoshpate, or IP3. IP3 then acts as a ligand to stimulate the release of Ca2+
from the endoplasmic reticulum into the cytosol. This released Ca2+ also acts as a second
messenger ligand by binding to a protein called calmodulin which becomes an active
messenger that can bind to other proteins to stimulate a cellular response, such as apoptosis or
cell death.

Cellular Responses to Signals: In response to a signal, a cell may regulate activities in the
cytoplasm or transcription in the nucleus. A signal may cause an ion channel to open (e.g.,
nervous or muscular systems) or a change in metabolism (e.g., epinephrine and glycogen to
glucose). Other signaling pathways stimulate genes to make proteins in response to a signal.
Elaborate pathways amplify and specify the cells response to signals. Signal Amplification may
occur at each step in transduction of a signal. This allows the cells response to be greater than
if there was no amplification, e.g., the binding of one epinephrine molecule can result in the
production of thousands of enzymes to hydrolyze glycogen into glucose.
The Specificity of Cell Signaling is due to different types of signal receptor proteins in plasma
membranes and different types of transduction proteins in the cytosol of a cell. Due to this, one
type of signal ligand can cause different cells to respond in different ways. Remember that
there must be ways to turn off each signal transduction pathway. Otherwise a cell would not be
able to respond to a new stimulus.

Anthrax (Bacillus anthracis)

Anthrax is an infectious disease caused by bacteria called Bacillus anthracis (shown above). The
bacteria are rod-shaped and tests gram positive when exposed to Gram stain, meaning it has an
exterior peptidoglycan layer. The cells are large with square ends and they have centrally
located spores. The bacteria can be grown in ordinary nutrient medium aerobically, or
anaerobically. The pathogenic effects of the bacteria are primarily caused by the fact that it
produces two types of toxins, lethal toxin and edema toxin, along with a protective capsule.
Anthrax is primarily a disease of domesticated and wild animals, particularly herbivorous
animals, such as cattle, sheep, horses, mules and goats. Humans become infected when
brought into contact with diseased animals, which includes their flesh, bones, hides, hair and
excrement.

Although the spores have been found naturally in soil samples from around the world, in the
United States there are only recognized areas of infection in South Dakota, Nebraska, Arkansas,
Texas, Louisiana, Mississippi, California and small areas that exist in other states. Even in
endemic areas, anthrax infections occur irregularly, often with many years between
occurrences.

In the United States, the incidence of naturally-acquired anthrax is extremely rare (1-2 cases of
cutaneous disease per year). Worldwide, the incidence is unknown, although B. anthracis is
present in most of the world. Unreliable reporting makes it difficult to estimate the true
Gram stained B. anthracis
Spores
incidence of human anthrax worldwide. However, in fall 2001, 22 cases of anthrax (11
inhalation, 11 cutaneous) were identified in the United States following intentional
contamination of the mail.
The most common form of the disease in humans is cutaneous anthrax, which is usually
acquired via injured skin or mucous membranes. A minor scratch or abrasion, usually on an
exposed area of the face or neck or arms, is inoculated by spores from the soil or a
contaminated animal or carcass. The spores germinate, vegetative cells multiply, and a
characteristic gelatinous edema develops at the site. This develops into papule within 12-36
hours after infection. The papule changes rapidly to a vesicle, then a pustule (malignant
pustule), and finally into a necrotic ulcer from which infection may disseminate, giving rise to
septicemia. Lymphatic swelling also occurs within seven days. In severe cases, where the blood
stream is eventually invaded, the disease is frequently fatal.
Another form of the disease, inhalation anthrax (called woolsorters' disease in the past),
results most commonly from inhaling spore-containing dust where animal hair or hides are
being handled. The spores can have a 7 42 day germination period, but when the disease
begins it is usually brought on suddenly with high fever and chest pain. It progresses rapidly to a
systemic hemorrhagic pathology and is often fatal if treatment cannot stop the invasive aspect
of the infection.

Gastrointestinal anthrax is similar to cutaneous anthrax but occurs on the intestinal mucus
lining. The bacteria spread from inflammation in the intestinal tract to the lymphatic system.
Intestinal anthrax results from the ingestion of poorly cooked meat from infected animals.
Gastrointestinal anthrax is rare but may occur as explosive outbreaks associated with ingestion
of infected animals, and has an extremely high mortality rate.
Clinical manifestations of Anthrax infection may include blood vessel leakage, pleural effusion,
hemorrhaging, sepsis and shock. Treatment is with antibiotics such as ciprofloxacin, which must
be administered immediately. The earlier treatment can be administered, particularly for
inhalation anthrax, the better the chances will be of recovery. Though not readily available,
people can be vaccinated against anthrax.

Dysfunction of Normal Cell Signaling By Anthrax
The virulence of B. anthracis, is due to the multicomponent anthrax toxins made up of PA
(protective antigen), LF (Lethal Factor), and EF (Edema Factor). PA binds to the surface of host
cells, allowing the LF and EF to enter.
The component, lethal factor (LF), is a metalloprotease, known to cleave most isoforms of
mitogen-activated protein kinase (MAPK)-kinases (MAPKKs) close to their N-terminus. This
interferes with a major signaling pathway linking the activation of membrane receptors, such as
toll-like receptors, to the transcription of several genes. Our normal cell signaling requires
kinases phosphorylating downstream substrates. LF prevents this by cleaving the MEK kinases,
thus stopping our normal cell signaling (by blocking downstream phosphorylation). This has
detrimental effects on our cells including: inhibiting cell growth, blocking phagocytosis,
stopping our adaptive immune response, along with cell death.
EF is a calcium-dependent adenylyl cyclase which converts ATP to cAMP. This leads to high
levels of cAMP inside our cells, causing an unregulated activation of the cAMP pathway. EF has
an overall inhibitory effect on our cells. These effects include: inhibiting microbicidal activity,
preventing phagocytosis, and blocking cytokine production.
LF gains access to the host cell cytosol after it is secreted by newly germinated spores
(vegetative bacteria, early infection) or after it is endocytosed from circulation during later,
systemic infection.

Sabotage of the Cell Signal

National Science Education Standards (1996)
Center for Science, Mathematics, and Engineering Education (CSMEE)

Standard: As a result of activities in grades 9-12, all students should develop abilities necessary
to do scientific inquiry and understanding about scientific inquiry. (Activity 1, 2, 3)
Understanding of scientific concepts.
An appreciation of "how we know" what we know in science.
Understanding of the nature of science.
Skills necessary to become independent inquirers about the natural world.
The dispositions to use the skills, abilities, and attitudes associated with science.

Standard: As a result of activities in grades 9-12, all students should develop an understanding
about science in personal and social perspectives. (Activity 1, 2, 3)
Natural and human induced hazards
Issues regarding personal and community health
Science and technology in local, national, and global challenges
Natural resources and environmental quality

Standard: As a result of activities in grades 9-12, all students should develop abilities of
technological design and understandings about science and technology. (Activity 1, 2, 3)
Abilities and understandings of technological design.
Creativity, imagination and a good knowledge base are required in the work of
engineering and science.
Scientists in different disciplines ask different questions, use different methods of
investigation, and accept different types of evidence to support these explanations.

Florida Sunshine State Standards

SC.912.N.1.1 (Activity 1 3): Define a problem based on a specific body of knowledge, for
example: biology, chemistry, physics, and earth/space science, and do the following:
1. pose questions about the natural world;
2. conduct systematic observations;
3. examine books and other sources of information to see what is already known;
4. review what is known in light of empirical evidence;
5. plan investigations;
6. use tools to gather, analyze, and interpret data (this includes the use of measurement in
metric and other systems, and also the generation and
interpretation of graphical representations of data, including data tables and graphs);
7. pose answers, explanations, or descriptions of events;
8. generate explanations that explicate or describe natural phenomena (inferences);
9. use appropriate evidence and reasoning to justify these explanations to others;
10. communicate results of scientific investigations; and
11. evaluate the merits of the explanations produced by others.

SC.912.N.1.6 (Activity 1,2,3) Describe how scientific inferences are drawn from scientific
observations and provide examples from the content being studied.

SC.912.L.14.2 (Activity 3) Relate structure to function for the components of plant and animal
cells. Explain the role of cell membranes as a highly selective barrier (passive and active
transport).

SC.912.L.14.6 (Activity 1,2,3) Explain the significance of genetic factors, environmental factors,
and pathogenic agents to health from the perspectives of both individual and public health.

SC.912.L.16.10 (Activity 1,2,3) Evaluate the impact of biotechnology on the individual, society
and the environment, including medical and ethical issues.

SC.912.L.18.10 (Activity 3) Connect the role of adenosine triphosphate (ATP) to energy transfers
within a cell.

SC.912.L.18.11 (Activity 3) Explain the role of enzymes as catalysts that lower the activation
energy of biochemical reactions. Identify factors, such as pH and temperature, and their effect
on enzyme activity.

HE.912.C.1.8 (Activity 2, 3) Analyze strategies for prevention, detection, and treatment of
communicable and chronic diseases.

LA.910.2.2.3 (Activity 1, 3) The student will organize information to show understanding or
relationships among facts, ideas, and events (e.g., representing key points within text through
charting, mapping, paraphrasing, summarizing, comparing, contrasting, or outlining).

Vocabulary

cAMP: cyclic adenosine monophosphate, a molecule derived from ATP and used as a secondary
messenger in cells for intracellular signal transduction.
Cytokines: small secreted proteins; important in mediating immune system and inflammation.
Conformational change: alteration in the shape, usually the tertiary structure of a protein, as a
result of alteration in the environment, pH, temperature, ionic strength or the binding of a
ligand (to a receptor) or binding of substrate (to an enzyme).Heterodimers (dimers): 2 separate
interacting polypeptide chains which are not the same.
Fibronectin: ECM glycoprotein; important in cell migration and wound healing.
GTPases (Rho family): GTP binding proteins
Integrins: superfamily of cell surface/transmembrane proteins that are involved in binding to
extracellular matrix components, most are heterodimeric (two amino acid chains).
Ligand: a substance that forms a complex with a biomolecule to serve a biological purpose. In a
narrower sense, it is a signal triggering molecule, binding to a site on a target protein.
MAPK/ERK (mitogen activated protein kinase or extracellular signal-related kinase) signal
pathway: Mitogen-activated protein kinase/extracellular signal-regulated kinase; a family of
serine/threonine kinases, which are phosphorylated by other kinases for full activity. Important
for gene transcription and regulation.
Phosphorylation: addition of a phosphate (PO43-) group to a protein or other organic molecule;
can activate or deactivate proteins.
Protein Kinases: phosphokinases, a family of enzymes that catalyze the transfer of phosphate
from ATP to a second substrate.

Additional Resources for Teachers:

Antibody Response Video clip
http://www.youtube.com/user/nucleusanimation

Atlas of Macromolecules, Protein Structure
http://www.umass.edu/microbio/chime/pe_beta/pe/atlas/atlas.htm

Cell Migration/Integrins
http://student.biology.arizona.edu/honors2001/group08/intro/intro1.htm

Western Blot Video Summary
http://www.biosolutions.info/2008/07/western-blot.html#

Western Blot Procedure Sheet
http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html

References

Todar, Kenneth PhD. 2011. Todars Online Textbook of Bacteriology.
http://www.textbookofbacteriology.net/Anthrax.html

Robinson, P; Fleming, E; Hack, C; Schneider, D; Gearhart, J (2010) "Biologically-Based Modeling
of Anthrax Infection: Modulation of Macrophage MAPK Signaling Pathway by Lethal Toxin",
JMedCBR 8, 4 September 2010,
http://www.jmedcbr.org/issue_0801/PJRobinson/PJRobinson_09_10.html.

Utah Education Resource: http://www.uen.org/Rubric/rubric.cgi?rubric_id=25

Images: Wikimedia commons images, or created by Melissa Guinta
http://commons.wikimedia.org/wiki/File:Anthrax_spores.jpg
http://commons.wikimedia.org/wiki/File:Bacillus_anthracis_Gram.jpg
http://commons.wikimedia.org/wiki/File:Anthrax_-_inhalational.jpg
http://commons.wikimedia.org/wiki/File:Anthrax_PHIL_2033.png
http://commons.wikimedia.org/wiki/File:Plastic_microcentrifuge_tube_rack-04.jpg
http://upload.wikimedia.org/wikipedia/commons/a/aa/200801large.jpg

Lesson 1: In the Eye of Infection (TEACHER Instructions)

Focus

Students participate in a jigsaw strategy to analyze a scenario about the effects of a bioterrorist
attack from the perspectives of government employees, healthcare workers, and laboratory
technicians.

Major Concepts

Biotechnology has an important impact in regards to the individual, society, and the
environment. When scientists are developing ideas, making inferences from observations and
the content available to them helps lead them in the right direction.

Objectives

After completing this activity, students will
Understand that scientific investigation affects public policy.
Recognize the flow of scientific investigation from observation to testing.
Understand the possible effects of Bacillus anthracis
Be able to organize information to show understanding or relationships among facts,
ideas, and events.

Prerequisite Knowledge

Scientific Method

Time for Activity

1-2 Class periods (~45 minute class periods)

Basic Science-Public Health Connection

This opening activity is to introduce your students to the complicated relationship between
government, scientists, and society and how these areas might interact during a health crisis. It
is also a way to introduce students to the way scientists must systematically study an issue
using the methods of science (for example, gathering and analyzing data).

Materials & Preparations:

Teachers will need to prepare the following materials before conducting this activity: (*based
on time available for activity)
Scenario 1: Narrator (per student)
Scenario 2: News Release (per student)
Scenario 3: Lab Personnel (1 for each student assigned this role) optional*
Scenario 3: Healthcare Administrators (1 for each student assigned this role)- optional*
Scenario 3: Government Employees (1 for each student assigned this role) optional*
Access to computer, internet, and projector for multimedia simulation on Anthrax
attack optional*

Procedure

DAY ONE
1. Students should be placed in groups of three. Each group is assigned one of the roles on
the following page.
2. Within the group, each student should receive a copy of Scenario 1. Using their own sheet
of paper folded hot dog style, instruct them to read the passage individually. After allowing
time to read the passage instruct students to discuss and record the important information
provided in the scenario for their assigned role in a column called What do you know on
the left side of the paper. Then instruct them to reflect and record information that would
be helpful to them in the assigned role, and record on the side labeled What do you need
to know.
3. Once completed, students are to be given scenario 2 and repeat the directions above to
read, discuss and record What do you know, and What do you need to know, from the
perspective of their assigned role.
4. Students should then be divided into groups containing one of each of the roles assigned.
There they are to take three minutes to each present their role and the information they
felt pertained to their role to the other two group members.
5. After each person has presented their perspective, students should be instructed to
brainstorm what information is needed to know for future action, and record a list of
options and suggestions for future investigations.

DAY TWO
1. Students should be placed in their groups of three, each with a distinct perspective
previously assigned. Provide students with Scenario Part 3 SPECIFIC TO THEIR ROLE. Give
students 15 20 minutes to read Part 3 and answer the questions that follow on their sheet
of notebook paper.
2. Have each group discuss what their questions were and how they answered, and make a list
of group suggestions for improving the response of the Federal Management Agency
emergency. (15 minutes)
3. Play the NTI BW Terrorism Tutorial: Go right to the link labeled Conclusions, and open the
class to a large group discussion comparing group suggestions to those provided on the site
above. (10 minutes)

http://www.nti.org/h_learnmore/bwtutorial/multimedia_02_02.html#pagetop
(Multimedia flash animation of an Anthrax attack.)

CDC/AMRIID,
LAB RESEARCHERS

LOCAL & FEDERAL
EMERGENCY
MANAGEMENT,
GOVT. OFFICIALS

HEALTHCARE
PERSONNEL, HOPITAL
ADMINISTRATORS

CDC/AMRIID,
LAB RESEARCHERS

LOCAL & FEDERAL
EMERGENCY
MANAGEMENT,
GOVT. OFFICIALS

HEALTHCARE
PERSONNEL, HOPITAL
ADMINISTRATORS

CDC/AMRIID,
LAB RESEARCHERS

LOCAL & FEDERAL
EMERGENCY
MANAGEMENT,
GOVT. OFFICIALS

HEALTHCARE
PERSONNEL, HOPITAL
ADMINISTRATORS

CDC/AMRIID,
LAB RESEARCHERS

LOCAL & FEDERAL
EMERGENCY
MANAGEMENT,
GOVT. OFFICIALS

HEALTHCARE
PERSONNEL, HOPITAL
ADMINISTRATORS

Scenario Part 1: (Narrator)

On the evening of November 1, a professional football game is being played a Southeastern
outdoor stadium before an audience of 74,000. The evening sky is overcast, the temperature
warm, a breeze blows from east to west. Two days after the game, hundreds of people in and
around the Southeast become ill with fever, cough, and in some cases shortness of breath and
chest pain.
Some of the sick self-administer over-the-counter cold remedies; some seek phone advice from
physicians and nurses; others are seen in clinics, doctors' offices, and emergency departments
throughout the city. Influenza cases had been seen in the Southeast 2 weeks before the game,
and health-care providers seeing the new patients recommend bed rest and fluids for the
presumed flu. Specimens are sent to confirm influenza. A few of the sickest patients get chest
radiographs to exclude pneumonia. A few patients are hospitalized; some have blood cultures
drawn.
The 400 ill persons in the region are receiving care from many different sources so a health
emergency is not detected. By November 4, nurses and physicians note the increased volume
of serious upper respiratory illness, and some contact the city health department for treatment
recommendations and a regional flu update. Blood cultures from the earliest patients grow
gram-positive bacilli in seven laboratories around the city. The laboratories identify these as
Bacillus species. No further identification is requested, and none is pursued.
By the third day after the game, patients with the earliest symptoms are dying. The illness has
been rapidly fatal, killing previously healthy young adults within 24 to 48 hours. Members of
the medical community, now alarmed by these unexpected and unexplained deaths, urgently
contact the state and city health departments. Health department officials contact the Centers
for Disease Control and Prevention (CDC). By midnight November 4, 1,200 people around the
city have fallen ill, 80 of whom have died.
[Patient sample case: A 63 year old man was taken by his wife to the emergency room with a
four day history of fever, myalgias, and malaise. His wife reported that he had a sore throat,
rhinorrhea, or other respiratory tract symptoms. He awoke confused and disoriented the
morning of admission. Medical history included mild hypertension and placement of a
coronary stent for atherosclerotic heart disease. He had also attended the football game
earlier in the week.]

What do you know? (From your assigned
perspective, re-read the above scenario and
list the information that is important for you
in your role)
What do you need to know? (List any
questions that relate to what is needed for
future decisions you may have to make in
your role):

Scenario Part 2 (News Broadcast)

"Previously healthy persons are dying of a rapidly fatal illness that spreads quickly among
health-care providers in the state. Expert consultants reached by CBS have suggested potential
diagnoses, including the new Spanish flu, Dengue Fever, Streptococcus, and many other
infectious and noninfectious diseases. A survey completed by city emergency departments and
health clinics finds that persons of all ages and from all sectors of the city continue to come
down with similar illness. The numbers have doubled since the previous day, inundating many
health-care facilities and straining an already decimated health care system.
In related News, an anonymous official has stepped forward to inform CBS that they have
knowledge that the Federal Bureau of Investigation (FBI) offices in five U.S. cities recently
received warnings of an imminent bioterrorist attack. Each threat indicated that a "shower of
anthrax would rain on U.S. cities," unless certain demands were met immediately. One of these
calls was reported to be to a large city on the Eastern Seaboard. The threats were credible, but
no information was relayed to city officials in Northeast or elsewhere. Could this be what is
affecting our city?"

What do you know? (From your assigned
perspective, re-read the above scenario and
list the information that is important for you
in your role)
What do you need to know? (List any
questions that relate to what is needed for
future decisions you may have to make in
your role):

Scenario Part 3: Government Response

The mayor convenes an emergency meeting of leading medical experts and health officials as
reporters gather outside city hall. Questions are how to proceed with the medical issues but
issues of the financial impact to the city from a major illness are also concerns. The
recommendations from all include isolation of all persons with fever, cough, or chest pain;
expanded laboratory analyses; and rapid epidemiologic investigation. How to implement the
recommendations are in question.
It is decided to send blood and tissue specimens to CDC for urgent analysis and CDC
investigators have been requested to arrive at the city as soon as possible. During a news
conference, the mayor describes the city's response to what appears to be a serious influenza
outbreak, appeals for public calm, and is surprised by questions about the possibility of
bioterrorism.
The mayor is outraged to learn that the FBI had not informed her of a credible anthrax threat to
the Southeast. She is informed that an anthrax vaccine exists, but it is unclear whether any will
be made available for civilian use. No one can yet estimate the probable scale of the epidemic
or whether there has been a single or multiple attacks. The antibiotic recommendations are
now being expanded to include all persons living or working within an area defined by 8 miles
west and 1 mile north or south of the stadium on November 1.
The mayor is told by her advisors that, in fact, no antibiotic arrivals are imminent. Some states
report they have no antibiotics to give, some are refusing to send shipments, and the federal
government reports that it will be at least another 6 hours before its antibiotic resources arrive.
Despite assurances that anthrax is not contagious, people with the ability to do so flee to the
North, causing traffic jams and increasing panic. Some train conductors, bus drivers, and pilots
refuse to transport, citing personal safety concerns and threatening to walk off the job if forced.
Businesses downwind of the stadium are shut down. The stadium is largely abandoned.
Newspapers brand the area "the dead zone."
The mayor holds a second press conference to address false allegations that anthrax vaccine is
being administered to select individuals in the city. She reports that federal authorities will
make available some vaccine for those deemed at highest risk. But due to a national shortage of
vaccine and military concerns that this attack may herald further attacks, there is only a highly
limited amount of vaccine available. For the most part, the city will have to manage with
antibiotics alone.
By midnight November 6, anthrax has sickened 3,200 people, 900 of whom have died.

Scenario Part 3: Hospital Personnel

The recommended isolation protocols quickly fall apart as hospital and clinic staffs struggle to
cope with the surge of patients. Fears of a contagious disease prompt hospital staff to don
protective positive-pressure hoods; the news shows physicians working in this gear and
explains that there are only two dozen or so such hoods available per hospital.
Hospitals around the city are then informed of the anthrax epidemic and warned to prepare for
a new surge of patients in the wake of the mayor's forthcoming TV address. Recommendations
for the care of infected patients are sent to hospitals and clinics around the region and they
outline that the state is seeking assistance from state and federal agencies. The mayor tells the
hospital staff that antibiotics must be taken by all those who attended the football game and
have come in for treatment. For those who attended the game and remain well, arrangements
are being made to distribute antibiotics at 20 police stations and schools around the city
starting immediately. The mayor wants healthcare personnel to stress that anthrax is not
contagious and appeals for calm.
By noon November 5, intensive-care units and isolation beds across the city are full. Even
patients receiving the most advanced medical care are dying. Patients are febrile, hypotensive,
and seem to be in septic shock; some have meningitis. Still, there is no diagnosis for some. At
some locations, the shock of rapid and unexplained deaths has created an atmosphere of
desperation and confusion among hospital and clinic staff. There are patients with cutaneous
infections (pustules on the skin), people with gastrointestinal anthrax infection from eating
food exposed to spores, and inhalation anthrax the most deadly form.
On the morning of November 6, the mayor announces that schools and homeless shelters will
be opened to the ill because hospitals can no longer accommodate new patients. The National
Guard will keep order. The Office of Emergency Preparedness, Department of Health and
Human Services, and the Federal Emergency Management Agency will provide some logistical
support.
Federal shipments of antibiotics have begun to arrive by November 7. The distribution centers,
now increased to 40, continue to be variably stocked with medicine. A heavy National Guard
presence is now evident at distribution centers to prevent violence. At this point, there are
effectively no antibiotics left in the city. Approximately 50,000 persons had obtained some
quantity before supplies ran out, but there is no record of who has received them. Health-care
facilities are unprepared to cope with the continually rising number of patients. By the early
hours of November 6, 2,700 persons have become ill with anthrax, 300 of whom have died.
Thousands more flood doctors' offices, clinics, and emergency departments, fearing that they
are infected with anthrax.

Scenario Part 3: Labs

In the early evening of November 5, a university laboratory makes a preliminary diagnosis of
anthrax from the blood culture of a young patient who died. The laboratory immediately
notifies city and state health departments, which in turn notify CDC and FBI. The specimen is
transferred to the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID),
where within hours experts report that rapid diagnostic tests support the preliminary diagnosis
of anthrax. CDC, FBI, and USAMRIID have stated that the working assumptions are that the
disease in Northeast is anthrax and that it is the result of a bioterrorist attack.
Widespread exposure to an anthrax aerosol is feared. CDC is seeking news of similar syndromes
in other locations around the country. The recommendations made to the mayor from the CDC
are that to prevent death, antibiotics must be given before symptoms occur, or at the latest, in
the earliest hours after symptoms begin. Patients with serious symptoms are likely to die, no
matter what anyone does. Available information suggests that the local supply of needed
antibiotics will soon be exhausted; many local pharmacies were already emptied of antibiotics
as the initial news of a lethal epidemic spread through the city.
Given this shortage of antibiotics, one senior advisor asks the mayor to consider a triage plan
that uses all available antibiotics to protect the exposed who are not yet sick. In this plan,
antibiotics would be kept from those already sick and thus likely to die, regardless of treatment.
The mayor requests immediate federal assistance in obtaining and distributing large supplies of
antibiotics. Antibiotic shipments from other states are also urgently requested. All that is
known is that many (but not all) of the dying had been at the football game on November 1.
At midday November 6, epidemiologists report that some anthrax patients had not attended
the game, suggesting that exposure had occurred over a wider area. In addition, computer
models show that wind patterns may have blown anthrax spores downwind of the stadium for
some miles. The antibiotic recommendations are now being expanded to include all persons
living or working within an area defined by 8 miles east and 1 mile north or south of the
stadium on November 1.
On televised interviews, families of the deceased promise legal action against the FBI for not
revealing the threats, and against local and federal government for not supplying sufficient
antibiotics and vaccine. Management of dead bodies becomes a growing crisis. Hospital and
city mortuaries are full. Many funeral homes have closed. The state health department and CDC
report that the deceased must be cremated. Some citizen and religious groups threaten that if
cremation is enforced, there may not be full reporting of the dead, and private burial
ceremonies would continue. By evening of Nov 7, a total of 4,800 persons have become ill;
2,400 have died.

Scenario 3 Questions

CDC/AMRIID/LAB RESEARCHERS
1. Should laboratory practices be changed to increase the chance of early detection of
anthrax?
2. Based on the information regarding the fact that antibiotics are in short supply and are
mostly not effective to those with severe symptoms, as a lab researcher, who do you
recommend receive them first?
3. Should an anthrax vaccine be more widely available (take into account issues of
production)?
4. How might health professionals and government officials interact with the media to
best inform the public and avoid misunderstanding and panic?
5. What are some of the additional health issues that scientists may worry about during a
crisis like this?

GOVT. AGENCIES
1. Could communities have plans for rapid mass antibiotic acquisition and distribution?
What are some of the problems they may have in doing so or doing without?
2. What should the community, hospitals, and professional societies be doing?
3. How might health professionals and government officials interact with the media to
best inform the public and avoid misunderstanding and panic?
4. Could outcomes have changed if state and local health officials had prior notification of
the anthrax threats?
mostly not effective to those with severe symptoms, as a government official, who do
you recommend receive them first?

HOSPITAL PERSONNEL
1. Should health-care workers become familiar with the early symptoms and signs of
anthrax?
2. Could outcomes have changed if state and local health officials had prior notification of
the anthrax threats?
mostly not effective to those with severe symptoms, as healthcare workers, who do you
recommend receive them first?
4. What are some recommendations healthcare personnel can suggest for the future?
5. How can they organize the health response within the hospitals?

Teacher Information: (The following is the original beginning of the scenario that may be used
for further discussion.)

On the evening of November 1, a professional football game is being played in Southeast's
outdoor stadium before an audience of 74,000. The evening sky is overcast; the temperature
mild, a breeze blows from west to east. During the first quarter of the game, an unmarked truck
drives along a highway a mile upwind of the stadium. As it passes the stadium, the truck
releases an aerosol of powdered anthrax over 30 seconds, creating an invisible, odorless
anthrax cloud more than a third of a mile in breadth. The wind blows the cloud across the
stadium parking lots, into and around the stadium, and onward for miles over the neighboring
business and residential districts. After the anthrax release, the truck continues driving and is
more than 100 miles away from the city by the time the game is finished. The anthrax release is
detected by no one.
Approximately 16,000 of the 74,000 fans are infected by the anthrax cloud; another 4,000 in
the business and residential districts downwind of the stadium also are infected. After the
game, the fans disperse to their homes in the greater southeast metropolitan area; some return
to homes in neighboring states. A few are from other countries. The driver of the truck and his
associates leave the country by plane that night. They will be many time zones away by the
time the first symptoms of anthrax appear 2 days later.
--------------------------------------------------------------------------------
Inglesby, Thomas V. Anthrax: A Possible Case History. Special Issue CDC Emerging Infectious
Diseases. http://www.cdc.gov/ncidod/EID/vol5no4/inglesby.htm Johns Hopkins School of
Medicine, Baltimore, Maryland, USA

Lesson 2: Identification of a Killer (TEACHER Instructions)

ELISA SIMULATION LAB

Focus

Students will continue learning from the scenario introduced in Activity 1, by completing an
ELISA simulation to diagnose whether those exposed to the Anthrax attack are exhibiting
symptoms from Anthrax and/or other possible pathogens.

Major Concepts

ELISA is commonly used to test blood serum for the presence of antibodies against disease
causing pathogens.

Objectives

understand the basic principles of antibody-mediated immunity
experience the steps involved in performing an ELISA
Understand how an ELISA is used as diagnostic tool by medical personnel
understand the disease-causing agent and transmission patterns of certain infectious
diseases


The body produces antibodies in response to an infection.

Basic Science-Public Health Connection

This activity introduces the use of the ELISA as a way of gathering data about a patients illness.
It exemplifies how scientific tests can be used to enhance public health.

Time to Implement

Approximately 45 minutes

ELISA Background Information

ELISA - Enzyme-Linked ImmunoSorbent Assay and is used in immunology to detect the presence
of an antibody or an antigen in a sample. It is often used as a diagnostic tool in medicine. In
our experiment today, we have known antigens from eight possible illnesses, fixed to the
surface of microtitration plates. When a serum produced from a patients blood is applied to
the plate, if there are antibodies present in for any of the illnesses being tested for the antibody
will attach to the matching antigen. After treatment with the antibody, additional reagents are
applied to enable a researcher to view the results. These reagents will react to where the
antibodies have attached and fluoresce when UV light is applied.
A similar procedure is possible using a plate pre-coated with an antibody which would react to
an antigen present in a patients blood serum. Either of these plate types can be prepared by a
researcher or purchased pre-made for repeated use.

Introduction

Eight patients have been sequestered in the local Health Department after expressing the
following symptoms: feeling light headed, sweating, coughing, malaise, and swollen lymph
nodes. Due to the nature of their symptoms and residence near the Football stadium which
was the site of the recent bioterrorist attack, an ELISA has been ordered to try and confirm
infection by the bacterium, Bacillus anthracis in a patients blood serum. ELISA test plates have
been prepared that have antigens for the following diseases pre-fixed in the assigned rows:
A. Anthrax
B. Pneumonia
C. Mononucleosis
D. Salmonella
E. Influenza
F. Streptococcus
G. West Nile
H. Dengue Fever

Materials Required For Lab:

64 microcentrifuge tubes
8 Racks/Holders
8 Pre-treated ELISA plates
16 patient serum samples
8 Pipets 20 ul, plus tips (If use disposable pipets: Need 80, 10 for each station)
Antigen
Coated
Plate
Antibody
Detection
Antibody
Flourogenic
Substrate
UV light
Paper towels
Food coloring
16 Test tubes
Permanent marker
16, 50 mL Beakers
1, 500mL Beaker
Dish detergent
2, 1000 mL beakers

To prepare the following simulated reagents for each group follow the instructions below:
Positive controls:
Make a 1 mL master solution of each and aliquot 100 ul of the master solutions into 8
microcentrifuge tubes
Label each top with a permanent marker with the abbreviations below.
Use water and a different color of food coloring to create reagent (suggested coloring
below).

Tween 20 Wash Buffer: Make a master solution of Wash Buffer using 1000 mL of water, 1
drop of blue food coloring, and 2.5 mL of dish detergent. Provide each group with a 50 mL
beaker of solution.
Fluorescent Tagged Antibodies: Make a master solution of reagent using 1000 mL of water,
1 drop of green food coloring, and 1 drop of yellow. Provide each group with a 50 mL
beaker of solution.
Patient Serum: Make a master solution of patient serum. Fill a beaker with 1000 mL of
water and add 1 drop of yellow food coloring. Label 16 test tubes with numbers 1-16 and
fill full with reagent. These are now your patient samples to be distributed with the
correct ELISA plate. For example, Plate 1: patient 1 and 2; Plate 2: patient 3 and 4 and so
on.

Anthrax
Pneumonia
Mononucleosis
Salmonella
Influenza
Streptococcus
West Nile
Dengue Fever

+An
+Pn
+Mn
+Sm
+Flu
+Stp
+WN
+Den
Red
Orange
Blue
Pink
Purple
Yellow
Green
Light blue
Laboratory Safety:

Gloves and goggles should be worn routinely throughout the experiment as good laboratory
practice.

Procedure

Step 1: Place 20 ul of each positive control into the correct well. Change tips or disposable
pipettes between different samples.

Step 2: Place 20 ul of distilled water in each of the negative control wells.
Step 3: Place 20 ul of the serum from your first patient into the wells 7,8, and 9, and note
patient number on data sheet.
Step 4: Place 20 ul of the serum from your second patient into the wells, 10, 11, and 12 and
note patient number on data sheet.

Step 5: Carefully place a small stack of paper towels on top of your ELISA plate and invert
quickly to dispose of solutions. Place the plate upside down on paper towels and tap a few
times to remove any remaining solutions.
Step 6: Add 20 ul of the Wash Buffer (mild detergent called Tween 20) to every well.

Step 8: Add 20 ul fluorescent tagged antibodies to each well. These antibodies will recognize
any antibodies from an unknown serum.

Step 9: Gently agitate plate on table to help facilitate binding.
Step 11: Place the ELISA plate under a UV light to check fluorescence and record results on
data sheet by shading in the circles that show up as positive after exposure.

Teacher Key for patient infection (ELISA plates)

Plate 1: Patient 1 = positive for exposure to Anthrax, Streptococcus.
Patient 2 = positive for exposure to Mononucleosis, Influenza
Plate 2: Patient 3 = positive for exposure to Salmonella, West Nile, Dengue Fever
Patient 4 = positive for exposure to Anthrax
Plate 3: Patient 5 = positive for exposure to Influenza, Steptococcus
Patient 6 = positive for exposure to Anthrax, Pneumonia
Plate 4: Patient 7 = positive for exposure to Anthrax, Influenza
Patient 8 = positive for exposure to Salmonella, Influenza, Streptococcus
Plate 5: Patient 9 = positive for exposure to Anthrax, Salmonella, Streptococcus, West Nile
Patient 10 = positive for exposure to Anthrax, Mononucleosis
Plate 6: Patient 11 = positive for exposure to Pneumonia, Mononucleosis, Salmonella,
Influenza, Streptococcus
Patient 12 = negative
Plate 7: Patient 13 = positive for exposure to Mononucleosis, Salmonella, Influenza,
Streptococcus
Patient 14 = positive for exposure to Anthrax, and Streptococcus
Plate 8: Patient 15 = positive for exposure to Influenza
Patient 16 = positive for exposure to Anthrax, Mononucleosis

Teacher Key for Lab Questions:

1. Why did you perform three identical tests for each control and patient sample?
Each assay was performed in triplicate to ensure reproducibility of the results.

2. What might cause some positive results to be lighter in color than others?
Weak positive results may be an indication that the patients blood serum carries few antibodies
against the disease-causing agent. The patients exposure to the pathogen may be recent and
the body may not have launched a full immune response yet. Alternatively, the infection may
have occurred long ago, and the level of antibodies in the patients bloodstream is declining.
(Note: Other explanations are possible and acceptable, but these are most likely.)

3. What is the function of the secondary antibody and fluorogen in an ELISA?
Antigen-antibody complexes formed in the initial steps of an ELISA are not visible to the unaided
eye. Therefore, a colorimetric detection system involving a secondary antibody and fluorogen is
employed. The secondary antibody, which is conjugated to an enzyme, recognizes and binds to
primary antibodies of antigen-antibody complexes, if they are present. Fluorogen substrate is
then added. If present, the enzyme linked to the secondary antibody attaches to the substrate
and causes it to fluoresce under UV light.

4. What basic principles of antibody-mediated immunity are utilized in an ELISA assay?
ELISA assays are based on the principles that antibodies are produced in response to infection
and that these antibodies are designed to specifically target particular antigens and bind tightly
to them.

5. Why do you think we used a negative control?
To ensure that our pre-treated plates would not react without antibodies applied.

6. Why might your patients have tested positive for more than one antibody?
More than one positive result may be an indication that the patients blood serum carries
antibodies against past disease-causing agents. The patients exposure to the pathogen may be
recent or as a child and the body may have launched a full immune response causing the
antibodies to still be present.

Lesson 2: Identification of a Killer (STUDENT Instructions)

ELISA SIMULATION LAB

Objectives:

understand the basic principles of antibody-mediated immunity
experience the steps involved in performing an ELISA
Understand how an ELISA is used as diagnostic tool by medical personnel
understand the disease-causing agent and transmission patterns of certain infectious
diseases

ELISA Background Information:

ELISA stands for Enzyme Linked ImmunoSorbent Assay and is used in immunology to detect
the presence of an antibody or an antigen in a sample. It is often used as a diagnostic tool in
medicine. In our experiment today, we have known antigens from eight possible illnesses,
fixed to the surface of microtitration plates. When a serum produced from a patients blood is
applied to the plate, if there are antibodies present in for any of the illnesses being tested for
the antibody will attach to the matching antigen. After treatment with the antibody, additional
reagents are applied to enable a researcher to view the results. These reagents will react to
where the antibodies have attached and fluoresce when UV light is applied.
A similar procedure is possible using a plate pre-coated with an antibody which would react to
an antigen present in a patients blood serum. Either of these plate types can be prepared by a
researcher or purchased pre-made for repeated use.

Introduction

Eight patients have been sequestered in the local Health Department after expressing the
following symptoms: feeling light headed, sweating, coughing, malaise, and swollen lymph
Antigen
Coated
Plate
Antibody
Detection
Antibody
Flourogenic
Substrate
nodes. Due to the nature of their symptoms and residence near the Football stadium which
was the site of the recent bioterrorist attack, an ELISA has been ordered to try and confirm
infection by the bacterium, Bacillus anthracis in a patients blood serum. ELISA test plates have
been prepared that have antigens for the following diseases pre-fixed in the assigned rows:
A. Anthrax
B. Pneumonia
C. Mononucleosis
D. Salmonella
E. Influenza
F. Streptococcus
G. West Nile
H. Dengue Fever

Materials

(Make sure you have the following materials at your station):
8 positive controls labeled for each disease (microcentrifuge tubes)
1 Rack/Holder
1 Pre-treated ELISA plate
2 patient serum samples
1 Pipette 20 ul, plus tips (If use disposable pipettes: Need 10 each station)
Access to a UV light
Paper towel stack

Laboratory Safety:

practice.

Procedure
Step 1: Place 20 ul of each positive control into the correct well. Change tips or disposable
pipettes between different samples.

Step 2: Place 20 ul of distilled water in each of the negative control wells.
Step 3: Place 20 ul of the serum from your first patient into the wells 7,8, and 9, and note
patient number on data sheet.
Step 4: Place 20 ul of the serum from your second patient into the wells, 10, 11, and 12 and
note patient number on data sheet.

Step 6: Add 20 ul of the Wash Buffer (mild detergent called Tween 20) to every well.


Step 8: Add 20 ul fluorescent tagged antibodies to each well. These antibodies will recognize
any antibodies from an unknown serum.

Step 9: Gently agitate plate on table to help facilitate binding.
Step 11: Place the ELISA plate under a UV light to check fluorescence and record results on
data sheet by shading in the circles that show up as positive after exposure.

Lab Questions: Complete the following on your own sheet of paper.

1. Why did you perform three identical tests for each control and patient sample?
2. What might cause some positive results to be lighter in color than others?
3. What is the function of the secondary antibody and flourogen in an ELISA?
4. What basic principles of antibody-mediated immunity are utilized in an ELISA assay?
5. Why do you think we used a negative control?
6. Why might your patients have tested positive for more than one antibody?

Data Sheet:
Shade the circles where color was detected after UV exposure.

Record your Patient #s and results below:

Patient _____: _________________________________________________

Patient _____: _________________________________________________

Lesson 3: Zooming In: (TEACHER Instructions)
What in the toxin is causing cell death?

Western Blot Analysis

Focus

In this experiment, students will use a modified Western Blot to test hypothetical cells from the
blood samples of patients with symptoms for Anthrax infection. Students are testing for the
presence of phosphorylated p38, which would show that the patients are not infected with
Anthrax. If the Anthrax lethal toxin is present in cells, it has been identified to block
phosphorylation of p38 MAPK (mitogen activated protein kinase) in the signaling pathway that
leads to macrophage activation. So instead of creating cells that can help defeat the infection,
the apoptosis of cells is triggered and rapid cell death occurs in patients.

Major Concepts

Western Blot analysis is used for analysis of proteins and can be used by medical personnel to
diagnose chronic infections. Cellular proteins are responsible for signal transduction and if not
able to function properly can lead to cell death.

Objectives

Understand the concepts and methodology involved with completion of a Western Blot
Analyze Western Blot Analysis as a strategy to detection and lead to treatment of
Understand that this technique is testing proteins because of their role in cell signaling


Students should be familiar with both macromolecule structure and function in the cell, and cell
signaling transduction pathways.

Basic Science-Health Connection

This activity incorporates information from Activity 1 and 2 and introduces the students to
more detailed techniques that researchers employ to assist doctors in understanding how an
illness is affecting the cell.

Time to Implement

3 or 4 - 45 minute class periods. May need adjustment

Introduction (provided for students)

Doctors in the scenario from Lesson 1 & 2 have sent a request to a CDC Lab to help them
understand how the Anthrax toxin is affecting their patients cells. They are seeking a
confirmation that the toxin is affecting cell signaling, in order to help guide them to potential
treatment options. They sent over new patient samples that they believe are positive for
Anthrax infection due to symptoms and they want the lab to test for the presence of
phosphorylated p38. The sample group of patient serum they provided consists of three
patients: 2 that were present at the football game where the bacteria was released, and 1 from
the surrounding area. All are believed to have Anthrax infection due to the symptoms they are
exhibiting. They believe that if non-phosphorylated p38 is present, this shows that the toxin is
blocking the phosphorylation of p38 protein in a kinase signaling pathway, and instead of
macrophage activation happening, this is the trigger for rapid cell death.

LABORATORY SAFETY:

practice.

MATERIALS NEEDED (For 6 lab groups)

EDVO-TEK KIT #275
Samples for Electrophoresis: Positive Control, Negative Control, 3 Patient Samples,
Standard Molecular Weight Dye Markers
UltraSpec-Protein Agarose
10x Tris-Glycine-SDS Buffer (Chamber Buffer)
10x Tris-Glycine Buffer (for gel preparation only)
Practice Gel Loading Solution
1 ml Pipets
100 ml Graduated Cylinder (packaging for samples)
Precut Western Blot Membranes (7 x 7 cm)
Precut Blotting Filter Papers (7 x 7 cm)
Western Blot Stain

ADDITIONAL MATERIALS NEEDED
6 Horizontal Gel Electrophoresis Apparatus
D.C. Power Supply (enough to run 6 gels)
Pipets with Tips
Microcentrifuge Tubes, 18-20
Beakers
12 Trays or Containers that can hold a 7 x 7 cm piece of membrane + 100 ml of liquid
Disposable Lab Gloves
Several Packs of Paper Towels
Plastic Wrap
Scissors
Metric Rulers
300 mL Methanol, 95-100%
6 , 50 mL graduated cylindars with tin foil as cover
25 mL Acetic Acid
1000 mL Distilled Water
6 pairs of Forceps
65 degree Celsius incubator
Glass tray for the stain

BACKGROUND INFORMATION: WESTERN BLOT ANALYSIS

Western Blot Analysis involves the direct transfer of protein bands from an agarose or
polyacrylamide gel to a charged nylon membrane for analysis. Following an electrophoresis
experiment, the gel is removed from the tray and the nylon membrane is placed directly on the
gel. Nylon membranes are much stronger and more pliable than gels and can undergo many
manipulations without tearing. Protein bands are transferred to the surface of the nylon
membrane and are adsorbed on the membrane by hydrophobic bonds. This transfer is achieved
electrophoretically in specially designed chambers, by capillary flow or by the application of
vacuum.

If samples are not prepared separately, the procedure for sample preparation would begin by
lysing the cells to release the proteins of interest. This solubilizes the proteins so they can
migrate individually through a separating gel. As soon as lysis occurs, proteolysis, de-
phosphorylation and denaturation begin. These events can be slowed down tremendously if
samples are kept on ice or at 4C at all times and appropriate inhibitors are added fresh to the
lysis buffer.
Antibodies typically recognize a small portion of the protein of interest (referred to as the
epitope) and this domain may reside within the 3D conformation of the protein. To enable
access of the antibody to this portion it is necessary to unfold the protein, i.e. denature it.
To denature, a loading buffer is used with the anionic denaturing detergent sodium dodecyl
sulfate (SDS), and the mixture is boiled at 95-100C for 5 minutes. Heating at 70C for 5-10
minutes is also acceptable and may be preferable when studying multi-pass membrane
proteins. These tend to aggregate when boiled and the aggregates may not enter the gel
efficiently.
SDS provides a uniform charge to all proteins so that they move through the gel based on size
only. When SDS is used with proteins, all of the proteins become negatively charged by their
attachment to the SDS anions. SDS denatures proteins by wrapping around the polypeptide
backbone.
SDS grade is of utmost importance: a protein stained background along individual gel tracts
with indistinct or slightly distinct protein bands are indicative of old or poor quality SDS.
The total protein transferred can then be visualized by staining the membrane with protein
dyes. Visualizing a specific protein within a mixture of proteins is usually detected by
immunochemical methods.

For immunological detection of a specific protein, the unstained membrane is placed in a
blocking buffer that contains detergent and protein that bind to all unoccupied sites on the
nylon membrane. The membrane is then incubated in buffer that contains antibody to one (or
more) of the blotted proteins. The antibody binds to the adsorbed protein (antigen) and
subsequent washings removes unbound antibody.

A second antibody that is covalently linked to an enzyme such as alkaline phosphatase or
horseradish peroxidase is used for detection.

The membrane is then incubated in a solution of the secondary antibody where it will bind
selectively to the bound antigen-primary antibody complex. Following this treatment, the
membrane is washed to remove the unbound secondary antibody-enzyme complex and is then
incubated in a solution containing a phosphatase or peroxidase substrate.
The products of the enzymatic reaction yield chromogenic (colored) products that are easily
visible on the nylon membrane.

PREPARATION OF AGAROSE GEL

1. To make 2.5% agarose in 1X gel buffer for a 7x7 cm size casting tray, add 0.5 gm of protein
agarose powder to 20 mL volume of buffer. Swirl to disperse clumps.
2. With a marking pen, indicate the level of the solution volume on the outside of the flask.
3. Heat the mixture to dissolve the agarose powder. The final solution should be clear (like
water) without any undissolved particles present.
a. Microwave method:
Cover flask loosely with plastic wrap to minimize evaporation. Do not cover
tightly.
Heat the mixture on High for 1 minute.
Swirl the mixture and heat on High in bursts of 25 seconds until all the agarose is
completely dissolved.

b. Hot Plate method:
Cover the flask with foil to minimize evaporation.
Heat the mixture to boiling with occasional stirring. Boil until the agarose is
completely dissolved.
4. Cool the agarose to 55C with swirling to promote even dissipation of heat. If detectable
evaporation has occurred, add hot distilled water to bring the volume of the solution up to
the original volume as marked on the flask.
5. After the agarose solution has cooled to 55C: Seal the ends of the gel tray with rubber
dams or tape. Seal the tray with a bead of agarose if tape is used.
6. Pipet the cooled agarose solution into the bed. Make sure the bed is on a level surface.
Place comb(s) in appropriate slots.
7. Allow the gel to solidify. It will become firm and ready for electrophoresis in approximately
20 minutes.

PREPARATION OF MEMBRANES
(Any time before the lab - required on DAY 1)
Wear rinsed and dried lab gloves. Powders from gloves will interfere with the procedure.

1. Keep protective cover sheets around the membranes and make sure the cover sheets and
membrane are all aligned. Keep the membrane covered this way during all the following
steps.
2. Divide the covered membrane into six 7 x 7 cm squares by drawing pencil lines on the upper
cover sheet. If you are using gels that are smaller or larger than 7 x 7 cm, you must adjust
the dimensions of your membrane squares accordingly. You may also have to alter the sizes
of the filter paper and towels the students prepare. Larger gels may necessitate fewer
groups.
3. Cut the covered membranes on the lines to produce six squares. Make sure the sheets are
aligned before cutting.

PREPARATION OF BUFFERS AND STAINS
(Required on DAY 1)

Transfer Buffer
1. To 350 ml of distilled water, add 50 ml of 10X Tris-Glycine-SDS concentrate.
2. Add 100 ml of 95 - 100% methanol to the buffer. Mix. Keep tightly covered.

Electrophoresis Buffer, Tris-glycine-SDS Buffer
1. Add 1 part EDVOTEK 10X Tris-Glycine-SDS buffer to every 9 parts distilled water.
2. Make enough 1X buffer for all electrophoresis units used. Most of the standard
electrophoresis units require between 300 - 400 ml (minimum) per unit.

PREPARATION OF SAMPLES FOR ELECTROPHORESIS
(Complete preparation on the day of the lab - Required first day)

1. Rehydrate the samples for electrophoresis (A-E) by adding 120 l of distilled water to each
tube. Incubate at room temperature for five minutes and vigorously mix. Use a water-
resistant marker to label the tops of the tubes A-E in case the labels come off during boiling.
2. Wear safety goggles, in case the covers pop off due to heat. Make sure the tube caps are
securely fastened. Suspend tubes A-E in a boiling water bath for 5 minutes. (It is not
necessary to boil the markers, Component F.) Remove and let them cool to room
temperature. Tap or briefly microcentrifuge to get condensate at the top of the tubes back
into the sample.

3. Aliquot 20 l of each sample (A-E) for each lab group. This experiment kit contains practice
gel loading solution. If you are unfamiliar with gel electrophoresis, it is suggested that you
practice loading the sample wells before performing the actual experiment. Do not use
Methanol with acrylic materials. Methanol will destroy acrylic.
Caution
PREPARATION OF STAIN SOLUTION
Make several days before the lab or on the day of the lab (required for DAY 3)

1. To 75 mL of distilled water, add 125 mL absolute methanol, 25 ml vinegar, and 25 mL
Western Blot Stain concentrate. Mix thoroughly.
2. Pour the stain into a glass tray. Do not use acrylic.

Day 1: Sample Preparation & Setting up the Electrophoresis.
Day 2: Setting up the Western Blot (Capillary Action)
Day 3: Processing and staining of the blot membrane, &bringing it all together.

Teacher Assessment

Lab report expanding on results and including future recommendations (graded by rubric).

Teacher Expected Results

A = Positive Control (phosphorylated p38) Normal cells = Positive/Band present
B = Negative Control (non-phosphorylated p38) Anthrax treated cells = No Band
C = Patient 1 (present at stadium) = Band Present/Negative for Infection
*D = Patient 2 (10 miles of stadium) = No Band Present/Positive for Infection
E = Patient 3 (present at stadium) = Band Present/Negative for Infection
F = Standard Molecular Weight Dye Markers

Students have shown that location and infection in this case are not necessarily related,
however, the correlation between the lack of phosphorylated p38 protein in infected cells can
be detected. A further confirmation of increased apoptosis in Patient 2 should be investigated.

Science Lab Report Rubric
Name: _______________________________

Excellent (20) Good (17) Satisfactory (15) Needs Improvement
(12)
Components of the Report All required
elements are
present and
additional
elements that add
to the report (e.g.,
thoughtful
comments,
graphics) have
been added.
All required
elements are
present.
One required
element is missing,
but additional
elements that add
to the report (e.g.,
thoughtful
comments,
graphics) have
been added.
Several required
elements are
missing.
Question / Purpose

Spelling, Punctuation, Grammar
The purpose of the
lab or the question
to be answered
during the lab is
clearly identified
and stated.

One or fewer
errors in spelling,
punctuation and
grammar in the
report.
The purpose of
the lab or the
question to be
answered during
the lab is
identified, but is
stated in a
somewhat unclear
manner.

Two or three
errors in spelling,
punctuation and
grammar in the
report.
The purpose of the
lab or the question
to be answered
during the lab is
partially identified,
and is stated in a
somewhat unclear
manner.

Four errors in
spelling,
punctuation and
grammar in the
report.
The purpose of the
lab or the question
to be answered
during the lab is
erroneous or
irrelevant.

More than four
errors in spelling,
punctuation and
grammar in the
report.
Drawings / Diagrams Clear, accurate
diagrams are
included and make
the experiment
easier to
understand.
Diagrams are
labeled neatly and
accurately.
Diagrams are
included and are
labeled neatly and
accurately.
Diagrams are
included and are
labeled.
Needed diagrams are
missing OR are
missing important
labels.
Error Analysis

Background Sources
Experimental
errors, their
possible effects,
and ways to
reduce errors are
discussed.

Several reputable
background
sources were used
and cited correctly.
Material is
translated into
student's own
words.
Experimental
errors and their
possible effects
are discussed.

A few reputable
background
sources are used
and cited
correctly. Material
is translated into
student's own
words.
Experimental
errors are
mentioned.

A few background
sources are used
and cited correctly,
but some are not
reputable sources.
Material is
translated into
student's own
words.
There is no
discussion of errors.

Material is directly
copied rather than
put into student's
own words and/or
background sources
are cited incorrectly.
Summary Summary
describes the skills
learned, the
information
learned and future
applications to real
life situations.
Summary
describes the
information
learned and a
possible
application to a
real life situation.
Summary
describes the
information
learned.
No summary is
written.
Appearance / Organization Lab report is typed
and uses headings
and subheadings
to visually organize
the material.
Lab report is
neatly
handwritten and
uses headings to
visually organize
the material.
Lab report is neatly
written or typed,
but formatting
does not visually
organize.
Lab report is
handwritten, looks
sloppy with cross-
outs, multiple
erasures and/or tears
and creases.

Lesson 3: Zooming In: (STUDENT Instructions)
What in the toxin is causing cell death?

Western Blot Analysis

Objectives:

Understand the concepts and methodology involved with completion of a Western Blot
Analyze Western Blot Analysis as a strategy to detection and lead to treatment of
Understand that this technique is testing proteins because of their role in cell signaling

Introduction:

Doctors in the scenario from Lesson 1 & 2 have sent a request to a CDC Lab to help them
understand how the Anthrax toxin is affecting their patients cells. They are seeking a
confirmation that the toxin is affecting cell signaling, in order to help guide them to potential
treatment options. They sent over new patient samples that they believe are positive for
Anthrax infection due to symptoms and they want the lab to test for the presence of
phosphorylated p38. The sample group of patient serum they provided consists of three
patients: 2 that were present at the football game where the bacteria was released, and 1 from
the surrounding area. All are believed to have Anthrax infection due to the symptoms they are
exhibiting. They believe that if non-phosphorylated p38 is present, this shows that the toxin is
blocking the phosphorylation of p38 protein in a kinase signaling pathway, and instead of
macrophage activation happening, this is the trigger for rapid cell death.

LABORATORY SAFETY:

practice.

PROCEDURE:

DAY ONE
Part 1: Sample Preparation
Take 5 of your microcentrifuge tubes and label them with permanent marker A E. Aliquot 20
ul of each of the reagents A E, that your teacher has prepared ahead of time, into each tube
and put on ice.
o A = Positive Control (phosphorylated p38) Normal cells
o B = Negative Control (non-phosphorylated p38) Anthrax treated cells
o C = Patient 1 (present at stadium)
o D = Patient 2 (from within 10 miles of stadium)
o E = Patient 3 (present at stadium)
o F = Standard Molecular Weight Dye Markers
Part 2: Setting up the Electrophoresis
You will need the following at your lab station:
o Protein Agarose 7x7 gel with wells (prepared by teacher)
o Approximately 350 mL of 10x Tris-Glycine-SDS Buffer (Chamber Buffer)
o 1, 10 ul micropipette with tips, or 8, 1 ml disposable Pipets
o Electrophoresis Horizontal Apparatus and Power Supply
o Samples from part 1
1. Remove 10 ul of sample A from the microcentrifuge tubes with your pipet.
2. Transfer the sample to the first well, taking care not to pierce the bottom of the well with
the micropipet tip. Do not overload the wells.
3. Repeat the above procedure from left to right with remaining samples. Make sure to
change tips on your pipet, or disposable pipets between samples!
4. Place your loaded gel, on a gel tray, in the center of the electrophoresis chamber. Position
the well side of the gel near the negative electrode.
5. Add approximately 350 mL of Tris Buffer to the chamber until the level of buffer is 2mm
above the top surface of the well.
6. Making sure that the cover and plugs are dry, slide the cover onto the electrophoresis
chamber (red on +, black on -).
7. Making sure that the patch cords attached to the cover are dry, connect the red cord to the
red electrode terminal on the power supply. Connect the black patch cord to the black
electrode terminal on the power supply.
8. ONLY NOW, plug the cord in and run at 125 volts, 45 minutes if possible, or until the blue
tracking dye has traveled at least 4 - 4.5 cm from the wells.
9. Unplug your apparatus, and carefully pour off buffer into waste container. Take out gel in
case and wrap snugly in plastic wrap for use on day 2.

DAY TWO
Part 3: Setting up the Western Blot (Capillary Action)
You will need the following at your lab station:
o Gel from Day 1
o 1 Precut Western Blot Membrane (7 x 7 cm)
o 2 Precut Blotting Filter Papers (7 x 7 cm)
o Western Blot Stain
o 20 mL of methanol in container with a cover
o 2 trays that can hold up to 100 ml of liquid
o Plastic wrap piece
o Distilled water
o Paper towels
o 50 mL Diluted transfer buffer
o Forceps
o 1 mL disposable pipet
o Pencil

1. Place a piece of plastic wrap on your bench top. Be sure it is smooth and flat. The filter
paper, gel, membrane, and paper towels will be placed onto it to make the blotting
sandwich.
2. Wearing gloves, carefully remove the cover sheets from the (white) Western Blot
membrane. Using forceps, transfer the membrane to a plastic tray.
3. Pre-wet the membrane (7 x 7 cm) by immersing it in approximately 20 mL of 95-100%
methanol for 10 seconds. Pour the methanol back in container to save.
4. Immediately pour 50 mL of distilled water onto membrane in tray and soak for about 5
minutes to remove the methanol (slightly agitate by sliding tray on table back and forth
gently).
5. Pour off the water and immerse the membrane in 20 mL of diluted transfer buffer. Let the
membrane sit until needed for the gel, at least 10 minutes.
6. Remove the gel from the plastic wrap and immerse into a separate tray that contains an
additional 25 mL of transfer buffer. Soak for 10 to 15 minutes.
7. Saturate 1 piece of 7 x 7 cm filter paper with transfer buffer by dipping it into one of the
trays. Place the filter paper on the plastic wrap on your bench.
8. Wear gloves and carefully remove the gel from the transfer buffer and place upside down
on the filter paper. Roll a pencil over the surface to remove air bubbles that may be trapped
under the gel.
9. Pipet 1 to 2 ml of transfer buffer over the top of the gel and place the Western Blot
membrane over the gel. Roll a pencil over the surface to remove air bubbles.
10. Use a pencil to lightly trace the location of each of the bands in lane 6 onto the membrane.
Beside each mark, indicate the color of the respective band. (B1 = Blue 1, B2 = Blue 2, P =
Purple, and R = Red).
11. Saturate 1 piece of filter paper with transfer buffer by dipping it into one of the trays, and
cover the membrane with the wet filter paper. Roll a pencil over the surface to remove air
bubbles.
12. Add a second piece of dry filter paper to the top of the stack. Remove air bubbles.
13. Evenly place a stack of 7 x 7 cm paper towels 4 to 6 cm in thickness on top of the stack.
14. Place a plastic tray or plate on top of the stack. Place a light weight beaker (400 ml size) on
top. Allow the protein transfer to proceed overnight or for a minimum of 4 hours.

DAY THREE
Part 4: Processing and staining of the blot membrane
o 2 pieces of Filter paper
o 20 mL of Methanol with cover
o 100 mL Distilled water
o Western blot stain
o 2 Trays
1. Remove the tray, paper towels and filter paper from the top of the membrane. Leave the
membrane in place on top of the gel.
2. Using a pencil, lightly trace the outline of each well and number according to loading
sequence onto the membrane. (Remember the gel is upside down.)
3. Using forceps, remove the membrane from the gel and place it on a clean piece of filter
paper. The side that was in contact with the gel should be facing up.
4. With a pencil, on one of the lower corners write F (for front). On the other corner, write
your group number or initials. Place the membrane in a 65C incubation oven for 10
minutes to fix the samples.
5. After 10 minutes, very briefly wet the membrane in methanol so that there are no dry
areas.
6. Transfer the membrane to distilled water for about 5 minutes.
7. Immerse the membrane in the Western Blot Stain for 10-15 minutes with occasional
agitation.
8. With forceps, transfer the membrane to a tray of Methanol and gently move the membrane
in the alcohol to de-stain by sliding the tray gently back and forth on the table. Look for the
positive control to appear. When the bands are evident, immediately transfer the
membrane to a tray of distilled water.
9. After several minutes, remove membrane from the distilled water, lay on a piece of filter
paper.
10. Compare the three patient results to the positive and negative controls.

Part 5: Setting a New Direction:
Write a lab report according to class protocol, following the rubric provided.
Research the following to include in your summary:
o Role of p38 in the cell
o Medications that may improve cell signaling
o Ways available treat anthrax victims
o Possibility of a vaccine production on a large scale
o Suggestions for future research on Anthrax effects

A B C D E F
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