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“Take Two and Call Me in the Morning”

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					 “Take Two and Call Me in the
         Morning”
A Case Study in Cell Structure and Function


                    by

            Peggy Brickman
           University of Georgia
   Part I - Mysterious Illness
• Well, Becky thought, being a dorm counselor for
  freshmen was not going to be that bad. She got a free
  room for the year and the food was plentiful - free
  steaks last week at an outdoor BBQ followed by a hay
  ride in a horse-drawn wagon in their welcome
  celebration.
• But, then again, it wasn’t perfect: she had ended up
  covered in bug bites; some of the students got sick
  from eating steak that was burned on the outside and
  raw in the middle; the horses had mucked up the
  courtyard; and pigeons had roosted on the dorm roof.
• At least tonight the students were finally settling in and
  quieting down, she mused.
                                                         2
          Part I, continued…
• The quiet was shattered a few minutes later when one
  of the other counselors, Ann, yelled through her door:
• ―Becky, we’ve got a problem. One of the students
  found a homeless kitten, and the girl has been
  keeping her in her room. I only found out because the
  girl, Ellie, just came to my room complaining of being
  sick. I felt sick too when I saw the mess that kitten
  made. I thought cats were born housebroken, but I
  guess not.‖
• ―Anyway, now I think Ellie might really be sick,‖ Ann
  continued. ―She’s feverish and says she’s going to
  throw up.‖
                                                          3
            Part I, continued
• ―What do you want me to do?‖ Becky asked.
• ―I’m freaking out!‖ Ann answered. ―Forget about the
  mess, just help me figure out what to tell them at the
  health center. I don’t know what she’s been exposed to.
  Or what we’ve been exposed to for that matter! This is
  the second girl this week with aches, fever, and
  nausea.‖
• ―My Mom sent me a bunch of medicine,‖ Becky
  answered. ―I’ll make a list of where we’ve been, what
  we’ve eaten, and what we’ve possibly been exposed to.
  Then we can start taking something right away to keep
  from getting it, too.‖
                                                     4
             Becky’s Task
•   The Health Center will be using the
    differences between organisms to diagnose
    and treat Ellie.
•   In the next 2 minutes, list the clues in the
    story that help you identify how Ellie could
    have contracted a disease with flu-like
    symptoms. Come up with possible
    suspects (organisms) that could cause her
    to be sick.
                                               5
                   Your Task
•   Becky did an Internet search and found 5 possible
    suspects that could be causing Ellie’s illness.
•   During this class session we will investigate the
    differences between them.
•   Organisms are usually distinguished by the
    characteristics you listed in your homework answers
    to Table 1. Add any details you missed so that
    when you hear the results of the Health Center tests
    you will be able to figure out what was making Ellie
    sick.
•   Fill in possible drug treatments in Table 2.

                                                        6
           Ellie’s Diagnosis
• Initial Identification: The Health Center collected
blood samples from Ellie and observed her cells
under a microscope. They identified foreign
structures with DNA and outer membranes. The
cells were gram negative and about 1/10 the size of
her cells.
• ―Ah, ha!‖ said Becky. ―That matches one of my
suspects. I knew those were a health hazard. I just
need to re-check the size thing. This internet chart
compares our cells to viruses and stuff.‖
                                                  7
                Metric Review
• 1 meter (m) = ~3 feet                         1 mm
                                         10-3
• 1 meter (m) = 1000                                   Cells like
  millimeter (mm)                        10-4            ours
• 1 millimeter (mm) = 1000
  micrometer (µm) (smallest              10-5




                                meters
                                                       Bacteria
  size distinguished by naked            10-6   1 µm
  eye)
• 1 micrometer (µm) = 1000               10-7          Viruses
  nanometer (nm) (only seen
                                         10-8          Proteins
  with light microscope)
• 1 mm poppy seed = (1000                10-9   1 nm   Atoms
  µm/mm) = 1000 µm

                                                             8
     Becky’s Internet Search
    Results – List of Suspects
Suspect 1: Coxiella
burnetii causes Q-fever.
Coxiella are often found in
livestock and are excreted in
milk, urine, and feces. Infection
occurs 2-3 weeks after
inhalation of barnyard dust.
They are 0.3-0.5 µm gram-
negative bacterium
(prokaryotes) that must invade
and reside inside human cells
to cause infection.
                                          9
                                    1µm
 Prokaryotes
• Unicellular
• Reproduce asexually
• Composition
  – Protected interior (cytoplasm) that
    contains genetic material (one circle of
    DNA) as well as complexes of protein
    enzymes to carry out necessary functions
    of gathering energy, manufacturing
    proteins (ribosomes), etc.
                                               10
  Prokaryotes
• Size
  – 0.2-10 micrometer (µm)
• Composition
  – Phospholipid membrane, many contain
    cell wall composed of peptidoglycan
    (positive for chemical Gram stain), those
    with little or no peptidoglycan called Gram
    negative (like Coxiella).
                                                  11
 CQ1: “That’s great,” Becky said. “My Mom sent
 me 3 different antibiotics to kill bacteria.”
 Given the description of Ellie’s test results,
 which antibiotic will definitely NOT work:

A: Amoxicillin, Penicillin, and other ß-lactams
   – Blocks the enzyme that normally creates links in
      peptidoglycan molecules.
B: Streptomycin
   – Blocks prokaryotic ribosomes.
C: Ciprofloxacin hydrochloride (Cipro)
   – Blocks bacterial DNA gyrase enzyme needed to
      counteract excessive twisting of DNA that occurs
      when circles of DNA are unwound to be copied
      into DNA or RNA.
                                                         12
CQ2: “Wait a minute!” Ann said. “The doctor
said the blobs in Ellie’s blood were 1/10th the
size of her cells. Could they be Coxiella?”

                              10-3   1 mm
                                            Cells like
  A: Yes                      10-4            ours

  B: No                       10-5
                     meters
                                            Bacteria
                              10-6   1 µm

                              10-7          Viruses
                              10-8          Proteins
                              10-9   1 nm   Atoms
                                                         13
  Part II: Microscope Analysis
Becky and Ann talked together outside the student’s room at
the student health center the next morning.
―You’re right!‖ Becky exclaimed after viewing photographs of
Ellie’s blood up close. ―I wish I hadn’t started taking the
antibiotics. The little crescent shaped structures that I thought
were the bacteria may not be. When you zoom in on them,
they show up clearly in the electron micrograph on the right.
They aren’t too big to be bacteria, but they aren’t too small to
be mitochondria or some kind of protozoan parasite.‖
―Wait a minute,‖ Ann replied. ―The things on the right are the
pathogens? Look at their insides, they can’t be bacteria.‖
―Why not?‖ Becky asked.                                      14
Part II, Continued




                 1µm
                       15
CQ3: “Well,” Becky admitted, “there should be differences
between Ellie’s cells and the little blobs they saw.
Otherwise, it might mean one of my other suspects is the
cause. These are some of the structures normally found in
all cells.”

“No,” Ann answered, “one isn’t.”
Which structure is NOT found in all cells?


         A: Cytoplasm
         B: DNA
         C: Outer phospholipid membrane
         D: Ribosomes
         E: Membrane-bound organelles

                                                   16
                  Eukaryotes Prokaryotes



       DNA        linear strands within           single circle in ―nucleoid
                membrane-bound nucleus                      region
       Size              5-100 µm                        0.2-10 µm

Organization   often multicellular, some have   usually single-celled,
                cell walls (no peptidoglycan) some have peptidoglycan
                                                      cell walls
 Metabolism    usually need oxygen to exist       may not need oxygen to
                                                           exist
 Organelles    membrane bound organelles          no organelles, different
                   like mitochondria                    ribosomes
  Examples     plants, animals, protists, fungi      bacteria, archaea
                                                                     17
       Becky’s Internet Search
      Results – List of Suspects

Eukaryote - Suspect 2:
Cryptococcus neoformans
2.5-10 µm encapsulated fungus found
in decaying pigeon or chicken
droppings. Inhaled as spores that
eventually spread to the brain causing
meningoencephalitis. Has a black
pigmented layer that can be seen
sometimes on bird seed.


                                         18
     Becky’s Internet Search
    Results – List of Suspects

Eukaryote - Suspect 3:
Toxoplasma gondii
• 4-6 µm single-celled
protozoan parasite of
mammals & birds.
• Most likely acquired through
ingesting cysts in
undercooked meat.


                                 19
       Becky’s Internet Search
      Results – List of Suspects
Suspect 3:
Toxoplasma gondii
• Usually no symptoms, but
can cause flu-like
complaints.
• Sexual life cycle occurs in
cats, so infection can follow
contact with cat feces.


• See QuickTime movies
―Invasion 4‖ and ―Escape 2‖ at
http://www.uvm.edu/microbiology/videos_ward.php?id=23   20
    Eukaryotes
• Uni- or multicellular.
• Reproduce asexually &
  sexually.
• Composition:
   – Genetic material (long linear strands of
     DNA chromosomes) especially isolated and
     enclosed in membrane (nucleus)
   – Some have cell walls (plants have
     cellulose, fungi ß-glucan)
                                                21
  Eukaryotes
• Size 10-100 (µm)
• Composition:
  – Phospholipid membrane outside, as
    well as inside.
  – Interior membranes separate
    functions such as gathering and
    transforming cellular energy and
    manufacturing macromolecules.
                                        22
Eukaryotic Organelles
  Animal Cell   Plant Cell




                             23
       Eukaryotic Organelles
Endoplasmic reticulum   Nucleus    Mitochondrion




     Golgi                        Chloroplast


                                                24
CQ4: Becky’s Anti-Eukaryotic Medicines:
   • Pyrimethamine, Sulfonamides: Interfere with enzymes
     used to make the folic acid needed to make thymine
     and uracil nucleotides.
   • Polyenes combine with a component of fungal and
     some bacterial membranes, disrupt and break them.
One of these drugs specifically affects one of the two
eukaryotic suspects. Which test of Ellie’s blood would help
you tell which eukaryotic suspect she was infected with?
      A: Presence of DNA.
      B: Presence of ß-glucan-containing cell walls.
      C: Presence of cellulose.
      D: Presence of peptidoglycan cell walls.
                                                       25
            Part III: Viruses
• Becky and Ann are back at the dorm waiting for the
  results of more tests.
• ―I’ve also got some tamiflu,‖ Becky volunteered. ―I
  mean, what if those cells in the picture aren’t really
  making her sick. Maybe she just has the plain old
  flu.‖
• ―What do you mean?‖ Ann asked. What’s the
  difference?‖
• ―Flu is a virus,‖ Becky answers. ―I’ve actually got
  two suspects that are viruses. They’re probably
  the most different from the prokaryotes and
  eukaryotes. They’re not even cells.‖
                                                    26
       Becky’s Internet Search
      Results – List of Suspects
                                       Electron micrographs
4.   Influenza Virus: Spread primarily
     through respiratory droplets from
     sneezing or coughing. Virus has
     single strand of RNA surrounded by
     phospholipids/protein envelope (80-
     120nm).
5.   West Nile Virus: Spread by
     mosquitoes that have previously
     fed on infected birds. 20% of
     infected people show symptoms.
     Single stranded RNA,
     phospholipid/protein envelope
     (50nm).
                                                       27
        Viruses

• Not cells
• Cannot reproduce alone
  – hijacks a host cell to replicate itself.
• Composition
  – Outer shell: repetitive protein often
    inserted into a lipid envelope (responsible
    for recognition and infection of host cell.)
                                                   28
         Viruses
• Size
  – Smallest Organisms (50nm)
  – 100 times smaller than bacteria
• Composition
  – Protected interior that contains genetic
    material (DNA or RNA) with important
    protein enzymes required for duplication.
                                                29
Virus
hijacking
host system




              30
Tamiflu: Blocks neuraminidase enzyme made by all influenza A
strains (cause the ―flu‖ and avian flu.) Viruses are unable to
remove sticky sialic acid, and can’t escape.




                                                        31
          Table 1
Take a few minutes to complete the
homework table comparing viruses,
bacteria, and eukaryotes.




                                     32
CQ5: Match the description with the suspect.
Use your responses to Table 1 to identify the
row below that best describes West Nile Virus.


    Circular Nucleus    Divides    Size    Cell     Sexual
     DNA               asexually           Wall   Reproduction
A      +        -         +        1µm      +          -
B      -       +          +        10µm     -          +
C      -        -          -       0.1µm    -          -
D      -       +          +        5µm      +          +



                                                           33
      Part IV: DNA Analysis
―Well, it isn’t viral,‖ Becky said, closing her cell
phone. ―No neuraminidase. But, they found some
foreign DNA with the sequence: AACGTGGTCGTT.
The closest match is a gene used to make
ribosomes (rDNA). They are searching a huge DNA
database of sequences to find the organism that has
the closest match.‖




                                                34
CQ6: Foreign DNA sequence isolated from
Ellie: ACGTGGTCGTT.
Which sequence is the best match with this
foreign DNA?

A: Ellie’s nucleus         ATGGTCTCAATG
B: Ellie’s mitochondria    TTGGTCCGTCAG
C: Coxiella bacteria       TTGGTCGGTCAG
D: Toxoplasma nucleus      AACGTGGTAGTT
E: Cryptococcus nucleus    ATGGTGGCAATG

                                             35
       Strange Similarities
―What’s weird is that Ellie’s mitochondrial DNA
matches the Coxiella sequence so closely.‖
Ellie’s mitochondria TTGGTCCGTCAG
Coxiella bacteria    TTGGTCGGTCAG
―That makes sense in a way,‖ Ann answered.
―There is a lot of evidence that points to
mitochondria being descendents of gram-negative
bacteria just like Coxiella.‖
―What type of evidence?‖ Becky asked.
                                                  36
Eukaryotic Organelles &
Endosymbiosis

prokaryote      N                  C        N


   Strange similarities:
   1. Chloroplasts and mitochondria are the same
      size as prokaryotes.
   2. Both have circular DNA without histones with
      similar sequence to photosynthetic bacteria
      (cyanobacteria) and obligate intracellular
      rickettsia bacteria.
   3. Both divide like prokaryotes.              37
Eukaryotic Organelles &
Endosymbiosis

prokaryote     N                  C       N


   Strange similarities:
   4. Have their own protein synthesis machinery
      (ribosomes) more like bacteria than
      eukaryotes (sensitivity to Streptomycin).
   5. Inner membrane of mitochondria contains
      unusual phospholipid characteristic of
      bacterial membranes.
                                              38
Similarities Used to Group
        Organisms




                         Single-celled eukaryotes
                         2 billion years ago
First prokaryotes
                                               39
3.75 billion years ago
CQ7: Which letter best defines the place
mitochondria would take next to their
closest relatives on this family tree of living
organisms?


 A
 B
 C
 D
 E

                                           40
       Finale: Ellie’s Prognosis
―Well, Ellie’s responding well to the pyrimethamines that the
doctors prescribed,‖ Becky commented to Ann while checking her
email a few days later at the dorm.
―Yeah, and we’re lucky the cipro we took couldn’t harm our cells,‖
Ann replied. ―We were so wrong! I’m never self-medicating again.
Do you think we should warn the other students. They might have
had contact with the kitten, too.‖
―We don’t know if it was from cat poop,‖ Becky answered. ―I
learned that something like 25-40% of American adults are
already infected with Toxoplasma gondii, and not because of their
cats—usually it’s from eating raw meat. Plus, apparently the
oocysts in fresh cat poop aren’t infectious for a couple of days.
So, if you scoop the box right away you don’t have to worry.‖
―So now I have to know how often the cat box is cleaned?! I don’t
think I’m cut out for this job!‖ Ann moaned.

                                                            41

				
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