Lab Exam Flashcards
What regulates the concentration of sugars (glucose) in the blood?
the antagonistic controls of insulin and glucagon
Which peptide hormone is released when glucose levels are high?
insulin
What releases insulin into the bloodstream?
the pancreas
What peptide hormone is released when glucose levels are low?
glucagon
What releases glucagon into the bloodstream?
the pancreas
what is the novel technique used in the Bo et al., (2013), study?
a technique that detects glucose concentrations using a DNAzyme (a synthetic enzyme)
What is DNAzyme?
a synthetic enzyme that detects glucose
made of a G-rich oligonucleotide and hemin
acts like a peroxidase in the presence of glucose oxidase and glucose to oxidase ADHP into resorufin
What is DNAzyme made of?
a G-rich oligonucleotide and hemin
What is the function of DNAzyme?
to oxidize ADHP into resorufin in the presence of glucose oxidase and glucose
acts like a peroxidase
What enzyme does DNAzyme imitate?
peroxidase
What is ADHP?
10-acetyl-3,7-dihydroxyphenoazine
What colour is ADHP before it’s oxidized?
colourless
What colour is ADHP when it’s oxidized?
red
How is the concentration of glucose determined?
spectrophotometry at 570 nm used to detect resorufin which a standard curve can be made from to determine glucose concentration in the sample
Who won the Nobel Prize in 1923? For what?
Frederick Banting won for discovery of insulin
What type of feedback loop is regulation of blood glucose?
negative feedback
explain how blood glucose levels are regulated by a negative feedback loop
when blood glucose is high:
- B cells of pancreas are stimulated to secrete insulin into the bloodstream
- insulin targets organs to increase their glucose uptake
= reduces glucose levels in blood (brings parameter back to set point)
AND
- a cells of pancreas are inhibited to prevent the secretion of glucagon into the bloodstream
= reduces glucose levels in blood (brings parameter back to set point)
Which pancreatic cells secrete insulin?
beta cells
Which pancreatic cells produce glucagon?
alpha cells
Where are the pancreatic cells which secrete insulin and glucagon located?
in the Islets of Langerhans of the pancreas
Describe how blood glucose levels are antagonistically controlled
insulin is secreted by pancreatic B cells to lower glucose levels
glucagon is secreted by pancreatic a cells to raise glucose levels
What was the purpose of the glucose metabolism experiment?
- to observe the behavioural differences (gill cover, movement) between control fish and fish exposed to insulin
- to use a DNAzyme to detect the concentration of glucose in the fish’s urine and blood samples when exposed to glucose or insulin, or neither (control)
What organism was used in the glucose metabolism experiment?
diamond tetras, Moenkhausia pittieri
What were the 3 parts of the glucose metabolism experiment?
A (lab coordinator): prepare hemin-G-quadruplexes DNAzymes solution
B: Observe fish behaviour (control v. insulin)
C: Measure glucose from tank water and blood samples (control v. glucose exposed v. insulin exposed)
What was the hemin-G-quadruplexes DNAzyme solution prepared?
- a purified G-rich oligonucleotide was mixed with a hemin solution (hemin, Tris-HAC pH 8, NaCl, Triton X-100)
- probe solution: ADHP + glucose oxidase (GOx) combined (ADHP + GOx + Tris-HAC pH 8, Triton X-100)
- oligonucleotide/hemin solution mixed with probe solution
How much insulin was the experimental fish exposed to to observe behaviour the glucose metabolism experiment?
0.5 IU/mL insulin in 200 mL of aquarium water at 25 C
What were the control conditions for the the glucose metabolism experiment?
200 mL of aquarium water at 25C
no additions
How were the data collected for behaviour observations in the glucose metabolism experiment?
for a period of 15s, a student counted the number of times the gill covers open and closed and another student counted the distance (cm) the fish moved, repeated this for 5 minutes
repeated the 15s interval counting for 5 minutes at 30, 60, 90, 120 minutes
at every half hour interval, samples of the aquarium water were taken
repeated for insulin-exposed fish
How were data collected for Part C (measuring glucose concentrations) in the glucose metabolism experiment?
spectrophotometry at 570 nm max absorption
How were the blood samples collected for the glucose metabolism experiment? How many were there per group?
12 blood samples were collected from the caudal vein of 3-5 fish and pooled and diluted by 100x
these fish included control, glucose exposed, and insulin exposed
How much glucose was the treatment fish orally administered for the blood samples in Part C of the glucose metabolism experiment?
1.67 mg glucose/g body weight
How much insulin was the treatment fish injected with for the blood samples in Part C of the glucose metabolism experiment?
0.01 IU/g body weight
How were the blood sample solutions prepared for spectrophotometry in the glucose metabolism experiment?
the 12 blood samples were combined with 1 mL of probe solution (ADHP + GOx + oligonucleotide/hemin)
What samples were used in the spectrophotometry part of the glucose metabolism experiment?
12 previously collected blood samples from control fish, glucose fish (1.67 mg glucose/g body weight), and insulin fish (0.01 IU/g body weight) + probe solution
and
the 8 aquarium tank water samples taken every half hour in Part B (contain urine with glucose levels) + probe solution
1 mL probe solution per sample = ADHP + GOx + oligo/hemin
10 uL tank water or blood per sample
How were the urine samples (tank water samples) and blood samples prepared for spectrophotometry?
10 uL of each blood and urine sample were combined separately with 1 mL of probe solution and incubated for 45 minutes
Why was urine and blood samples used in the spectrophotometry for the glucose metabolism experiment?
glucose levels in urine are a good indicator for the amount of glucose in the bloodstream (high in urine = high in blood)
blood is a direct indicator for how much glucose is in the bloodstream
What maximum absorbance was the spectrophotometer set to for the glucose metabolism experiment?
570 nm
What was the blank solution used for the spectrophotometry in the the glucose metabolism experiment?
the probe solution (ADHP + GOx + hemin/oligo)
Why was a standard curved used for the assessment of glucose concentrations in the samples for the the glucose metabolism experiment?
a standard curve shows a linear relationship between a concentration of an unknown solution in a sample and its corresponding absorbances at 570nm
the equation of the line can be used to determine the glucose concentration for each sample
What must be considered when determining the glucose concentrations from the standard curve for the the glucose metabolism experiment?
the dilutions made before blood samples were mixed with probe solution (100x)
What trends were observed in the urine-glucose experiment for the glucose metabolism experiment?
glucose concentration in urine per gram of fish was higher in the insulin-exposed fish than in the control
unexpected - additional insulin should lower urine-glucose until glucagon production can catch up
reasons?
- maybe not enough time for glucose to metabolize?
- maybe too much time and glucagon caught up and produced more glucose?
- type of insulin used?
- dose?
- location of injection?
- timing and content of last meal?
What trends were observed in the blood-glucose experiment for the glucose metabolism experiment?
blood from fish exposed to glucose had highest glucose concentrations
blood from fish exposed to insulin had lowest glucose concentrations
blood from control fish had middle ground glucose concentration
expected results: additional glucose should raise blood-glucose until insulin production can catch up; additional insulin should lower blood-glucose until glucagon can catch up; in the control, glucagon and insulin secretion should be balanced
What trends were observed in the gill cover movement experiment for the glucose metabolism experiment?
insulin-exposed fish had higher rate of gill cover movement than control
explanation: possibly because an increased insulin content will increase the rate of glucose metabolism = increase the demand for O2 = respiration occurs over the gills in fish so more movement required to pass more oxygenated water across gills
What is spectrophotometry?
a technique used to quantitatively measure energy (usually light) absorbance by a specific molecule in a solution
can be used to determine a molecule’s concentration in a solution
What is the maximum absorption?
the wavelength (nm) that light is being maximally absorbed by the specific molecule in the solution
What is the relationship between absorbance and the concentration of the molecule in the solution?
they are proportional to each other
and they are proportional to the length of the light path
What is Beer’s Law (or Beer-Lambert Law)?
Absorbance of a molecule is proportional to its concentration in the sample and to the length of the light path
A = kLC
A = absorbance (no units)
k = coefficient of wavelength-dependent molar absorptivity (1/M*cm)
L = length of light path (cm)
C = concentration of absorbing molecule (M)
Why does absorbance have no units?
it’s a ratio of the intensity of the light hitting the absorbing molecule to the amount of light that’s passing through the absorbing molecule
all the units cancel each other out in A = kLC
What is Intensity (I) in spectrophotometry?
the amount of electromagnetic radiation occurring on a solution
ie., the energy of radiation per unit area per unit time
is this the amount of light hitting the solution?
What is transmittance in spectrophotometry?
the %T is the amount of light that hits a solution that actually passes through the solution
What does it mean for a solution to have %T = 100?
the solution is completely transparent and 100% transmittance is occurring
ie., 100% of the light hitting the solution is also passing through the solution
What is the equation for %T?
%T = 100(It / I0)
where
%T = transmittance
It = light transmitted through the sample
I0 = light hitting the sample
What is wavelength in spectrophotometry? what is it based on? how is it often represented?
represented symbolically by lambda
the frequency at which a molecule is absorbing electromagnetic radiation
based on %T
represented logarithmically because of the massive range of values
How is the absorbance of a sample represented (what is the formula)?
A = log(100 / %T)
or
A = 2.000 - log (%T)
2.000 because log(base10) * 100 = 2
What will the absorbance of a transparent sample be?
when %T = 100, A = 0
A = log(100/100) = 0
What will the absorbance of a sample be when %T = 10?
When %T = 10, A = 1
A = log(100/10) = 1
What will the absorbance of a sample be when %T = 1?
A = log(100/1) = 2
What will the absorbance of a sample be when %T = 0.1? %T = 0.01?
A = log(100/0.1) = 3
A = log(100/0.01) = 4
Describe a standard curve in spectrophotometry
absorbances for the standards are plotted as a function of their concentrations
it assumes linear relationship between concentration and absorbance
uses a control that contains all the reagents except the unknown
What is the control in spectrophotometry? what was it in the glucose metabolism experiment?
the blank solution - contains all reagents except for the one with unknown concentration
in the experiment, it was the probe solution (ADHP + GOx + oligo/hemin)
How is the blank used to determine the maximum absorbance?
set the spectrophotometer to 0 and insert the blank, measure the max absorption wavelength and use this to measure the wavelengths for the samples
How can the equation of the line produced by the standard curve be used to determine concentrations of glucose?
the absorbances of each sample are plotted against the concentrations and the slope is used to calculate the unknown concentration
x = kL
where
k = y/x (slope)
y = absorbance
x = unknown concentration
What 4 animal types were used in the comparative anatomy lab?
fish
adult frog (amphibian)
bird (reptile)
mouse (mammal)
What is positive pressure breathing?
when the pressure inside the lungs is greater than the pressure in the atmosphere
what is negative pressure breathing?
when the pressure inside the lungs is lower than the pressure in the atmosphere
What type of integuments do each of the 4 animal types have?
fish: skin + scales
frogs: skin
birds: skin + feathers
mouse: skin + hair (fur)
Are mucous cells present in each of the 4 animal types?
fish: yes
frogs: yes
birds: no
mouse: no
Is thermoregulation exhibited in each of the 4 animal types?
fish: no, ectotherm
frog: no, ectotherm
bird: yes, endotherm
mouse: yes, endotherm
What appendages do each of the 4 animal types?
fish: paired and unpaired fins
frogs: tetrapod, paired fore and paired hind limbs
birds: paired wings, paired hind limbs
mouse: tetrapod, paired fore and paired hind limbs
How many circulatory circuits do each of the 4 animal types have?
fish: 1
frog: 2
bird: 2
mouse: 2
How many heart chambers do each of the 4 animal types have?
fish: 2
frog: 3
bird: 4
mouse: 4
What are the respiratory organ(s) used in each of the 4 animal types?
fish: gills
frog: skin + lungs
bird: lungs
mouse: lungs
Are air sacs or alveoli present in each of the 4 animal types?
fish: no (no lungs)
frog: yes
bird: yes
mouse: yes
What are the gas exchange surfaces in each of the 4 animal types?
fish: lamellae of gill filaments
frog: skin + alveoli in lungs
bird: parabronchi in lungs
mouse: alveoli in lungs
Are the air sacs or alveoli directly involved in gas exchange in each of the 4 animal types?
frog: NA (no air sacs or alveoli, no lungs)
fish: yes
bird: no (parabronchi instead)
mouse: yes
What type of pressure breathing do each of the 4 animal types have?
fish: NA (no lungs)
frog: +
bird: +
mouse: -
How do the respiratory systems compare between fish and frogs given that they’re both aquatic?
fish:
- organ: gills
- gas exchange surfaces: lamellae of gill filaments
- dissolved O2 from water
- countercurrent, unidirectional flow of O2
frog:
- organ: skin + lungs
- gas exchange surfaces: skin + alveoli in lungs
- O2 from atmosphere
- tidal flow bidirectional through lungs
- non-directional flow over skin
fish do not have lungs, therefore do not have alveoli or any type of pressured breathing
frogs have lungs with alveoli for gas exchange and have + pressure breathing
How do the circulatory systems compare between fish and frogs given that they’re both aquatic?
both are closed systems with a chambered heart pumping blood through vessels
fish:
- 2 chambers (1 atrium + 1 ventricle)
- single circuit
frog:
- 3 chambers (2 atria + 1 ventricle)
- double circuit (pulmonary + systemic)
How do the digestive systems compare between fish and frog given they’re both aquatic?
How does the respiratory system compare between frog and mammals given they’re both tetrapods?
both have lungs with alveoli and have tidal bidirectional ventilation, but frogs can also respire across their skin
frogs:
- organ: skin + lungs
- GE surface: skin + alveoli in lungs
- both tidal bidirectional (lungs) and nondirectional (skin)
- ectotherm (metabolism lower = respiration lower?)
mammals:
- organ: lungs
- gas exchange surface: alveoli in lungs
- only tidal bidirectional ventilation
- endotherm - metabolism higher (respiration higher?)
How does the circulatory system compare between frog and mammals given they’re both tetrapods?
both have closed circulatory systems with a chambered heart to pump blood through vessels through TWO circuits
frog:
- 3 chambered heart (2 atria + 1 ventricle)
mammal:
- 4 chambered heart (2 atria + 2 ventricles)
How does the digestive system compare between frog and mammals given they’re both tetrapods?
How does the circulatory system compare between birds and mammals given that they’re both endotherms?
both have double circuit closed circulatory systems with a 4 chambered heart to pump blood through the vessels
- both have hepatic portal systems
birds:
- aortic arch arches to the right
mammals:
- aortic arch arches to the left
How does the respiratory system compare between birds and mammals given that they’re both endotherms?
Both have lungs as their respiratory organ
birds:
- GE surfaces: parabronchi in lungs (alveoli not involved in GE)
- crosscurrent unidirectional ventilation (2 exhale, 2 inhale)
- highly efficient at GE
- continuous
mammals:
- GE surfaces: alveoli in lungs
- tidal bidirectional ventilation
How does the digestive system compare between birds and mammals given that they’re both endotherms?