Exam 1 Week 3 Flashcards
(134 cards)
1
Q
How does Irreversible Inhibition form? How can this be overcome?
A
- Covalent bonding
2. Must synthesize a new enzyme that isn’t inhibited yet
2
Q
What 3 things do irreversible inhibitors cause?
A
- Covalent Mod of the Active Site
- Covalent bonding to -SH (Cysteinyl residues)
- Covalent interference with Metal CoFactors
3
Q
Give an example of an irreversible inhibitor and how it works:
A
DFP (Di-Isopropyl Fluorophosphate: “Nerve Gas” that inhibits AcetylcholineEsterase (Normally degrades ACh, leads to continuous stim of ANS)
4
Q
Give an example of a beneficial irreversible inhibitor:
A
Aspirin: An NSAID that inhibits COX (CycloOxygenase) which normally synthesizes Prostaglandins and Thromboxane (Needed for clotting blood)
5
Q
Describe DFP:
A
- Di-Isopropyl Flurophosphate
- Nerve Gas that inhibits IRREVERSIBLY Acetylcholinesterase, so ACh remains in the NMJ and overstimulates muscles
- Prevents Serine from covalently bonding with Acetyl group
6
Q
Describe Aspirin:
A
IRREVERSIBLE inhibitor of COX1/2 which is needed for thromboxane and prostaglandin synthesis to clot blood
7
Q
In what cells does Aspirin inhibit COX1/2 and why?
A
- Platelets and Endothelial Cells
- Platelets use COX to make Thromboxane, which clots blood BUT when inhibited they CAN’T make more COX, so no more Thromboxane is made.
- Endothelial cells use COX to make Prostacyclin, which reduces clotting AND when inhibited they can just make more COX so Prostacyclin is still released.
8
Q
Describe “Suicide Inhibitors” and give an example:
A
- Drugs that looks just like the substrate (like competitive inhibitors) but once bound, produce a structural modification that irreversibly inhibits the enzyme.
- Allopurinol
9
Q
How specifically does Allopurinol conduct its inhibition reaction?
A
It binds to Molybdenum-Sulfide, a CoFactor of the enzyme Xanthine Oxidase in the active site
10
Q
4 Methods of Regulation of Metabolism:
What does each one cause?
A
- Substrate/Product Concentration:
a. ) Increased Substrate = Increased Vmax
b. ) Increased Product = Decreased Enzyme Activity - Enzyme concentration:
a. ) Induction/Repression of transcription by hormones
b. ) Degradation of enzyme - Enzyme Modification:
a. ) Phosphorylation/De-phos. (reversible)
b. ) Proteolytic Activation (irreversible) - Allosteric Regulation:
a. ) Doesn’t follow Michaelis-Menten Kinetics, produces a sigmoidal curve.
11
Q
Give an example of an enzyme regulated by each of the 4 regulation methods:
A
- Product Concentration –> Hexokinase (neg. feedback)
- Enzyme Concentration –> HMG-CoA Reductase (pushed out of membrane by chol. and degraded when too much of it)
- Enzyme Modification –>
a. ) Reversible = Glycogen Synthase (de-phosphorylated when Inulin is high, WHICH ACTIVATES IT
b. ) Irreversible = Trypsinogen (truncated by enteropeptidase and becomes permanently active) - Allosteric –> Hb has cooperative binding, oxygen acts as an allosteric regulator that increases it’s affinity for more oxygen when bound by it.
12
Q
What does K-0.5 represent?
A
The Km of Allosteric enzymes
13
Q
List the 3 databases where genetic information is stored:
A
- NCBI: National Center for Biotechnology Information
- EMBL: European Molecular Biology Lab
- DDBJ: DNA Data Bank of Japan
14
Q
Describe the action of Restriction Endonucleases:
A
- Recognize and cut DNA in a specific and reproducible manner.
- Hydrolyze phosphodiester bonds leaving STICKY ENDS: A free 3’-OH and a 5’-Phosphate
15
Q
Which restriction endonucleases do we focus on and what do they do?
A
- Class 2 Restriction Endonucleases
- Homodimer enzymes that have one component recognizing one sticky end, and another component recognizing the other sticky end to give SYMMETRICAL, PALINDROMIC sequences.
16
Q
List the 4 types of DNA Polymerases focused on in lab studies:
A
- DNA Polymerase 1
- Klenow Fragment DNA Polymerase
- Reverse Transcriptase (RNA-dependent)
- Taq Polymerase
17
Q
What is DNA Polymerase 1 used for and why?
A
- Probe Prep and Making blunt ends from sticky ends
- It has 3 functions:
a. ) 5’-3’ Polymerase
b. ) 5’-3’ Exonuclease
c. ) 3’-5’ Exonuclease
18
Q
What is Klenow Fragment DNA Polymerase used for and why?
A
- Sanger Sequencing and cloning (cDNA production)
- It uses an ssDNA template and has NO 5’-3’ Exonuclease activity, only 5’-3’ Polymerase and 3’-5’ proofreading exonuclease.
19
Q
Compare Klenow Fragment DNA Pol. and Reverse Transcriptase?
A
Klenow needs an ssDNA template, Reverse Transcriptase needs an RNA template
20
Q
Describe Taq Polymerase:
A
- ONLY has 5’-3’ Polymerase activity
2. Requires Primers, used in PCR
21
Q
Why is Ligase important in lab studies?
A
Any piece of DNA cut by restriction enzymes will have the SAME STICKY ENDS and therefore can be joined together –> Creates RECOMBINANT DNA
22
Q
Describe Cloning:
A
- Use restriction endonucleases to cut a fragment of DNA into a specific size, and electrophoresis to separate those fragments by size.
- DNA Ligase will ligate it into a plasmid (circular dsDNA) vector.
- Insert it into “host” replicating cells.
- Allow the host cell to integrate, and replicate, this foreign DNA EACH TIME IT DIVIDES
(So you make tons more of it)
Fundamental to making Recombinant DNA
23
Q
What was the first widely used cloning vector? Explain the general concept of how cloning vectors work:
A
- pBR322
- They possess only ONE OriC, so we know exactly where the restriction sites are for Ampicillin.
- So we grow them in cells treated with a selectable marker (ampicillin), and only the cultures with the plasmid DNA vector will proliferate.
24
Q
List the 3 Important Features of Cloning Vectors:
A
- Able to self-replicate
- Have a Multiple Cloning Site: A number of unique restriction sites that are all close together and don’t present anywhere else in the vector (identifiable).
- Contain a Selectable Marker: A gene which gives them Antibiotic resistance, and therefore gives their host cell that resistance as well.
25
3 Things Cloning Vectors are used for:
1. Replication of target DNA
2. Sequencing: Since they have universal primers
3. Protein Expression: Since their promoter sequences can induce expression of eukaryotic genes once integrated.
26
Describe PCR:
1. Heat DNA to denature/separate into ssDNA
2. Add primers for top and bottom ssDNA strands, and allow sequence to cool so primers can anneal to the sequence of interest (since primers have 3'-OH)
3. Bring to optimum temp. for Taq Polymerase to allow DNA synthesis.
4. Repeat many, many times
27
Why is Taq Polymerase used in PCR (2 Reasons):
1. It only has 5'-3' Polymerase activity
| 2. It is THERMOSTABLE
28
Give the 2 things Taq Polymerase requires to function:
1. Primers (3'-OH)
| 2. ssDNA template
29
List the 6 things required for PCR to occur:
1. Taq Polymerase
2. ssDNA template
3. Primers
4. dNTP's
5. MgCl Cofactor for Polymerase
6. Buffer: To ensure optimal reaction conditions
30
How are primers designed?
1. To be complimentary to the FLANKING REGIONS of the target sequence in each of the two ssDNA templates
2. Forward Primer = Complimentary to the 3' end of the target sequence in the BOTTOM strand
Reverse Primer = Complimentary to the 3- end of the target sequence in the TOP strand
31
Describe how Coding DNA (cDNA) is made:
1. Isolate mRNA by targeting Poly-A tail
2. Add Oligo-dT (Thymine) Primers to bind tail
3. Add dNTP's and Reverse Transcriptase to reverse transcirbe the mRNA FROM THE Poly-T tail (5' END)
4. Destroy the mRNA by cleaving it with Alkali and use the new cDNA strand as template by adding DNA Pol. 1 to remove the mRNA fragments and replace them with DNA.
5. Add DNA Ligase to re-anneal the fragments and complete the new dsDNA sequence.
32
After cDNA is made, what is it used for?
PCR: Add Taq Polymerase, primers, dNTP's and use cDNA as the template to be amplified (after denaturing the dsDNA to give two ssDNA sequences for PCR)
33
What is typical seen in the first lane of an agarose gel used in electrophoresis?
"Molecular Weight Control": Samples of each fragment size and where it should be located between the +/- poles to compare against the other lanes.
34
What is a probe?
A segment of ssDNA designed to be complimentary to the KNOWN sequence of interest in genomic DNA and tagged with either RADIOACTIVE or FLUORESCENT labels.
i.e. If it hybridizes (after denaturing the genomic DNA to become ssDNA) then we know the patient possesses the sequence of interest. If not, they have a mutation or don't possess that sequence.
35
Describe how Southern Blot works:
1. Digest DNA with restriction endonucleases
2. Perform electrophoresis to separate by fragment size using Ethidium Bromide (alkaline) solution
3. Transfer this gel to a photo-sensitive film/membrane
4. Hybridize a radioactively labeled probe to this film
5. Wash off excess probe (unbound)
6. Expose film to X-ray to see which fragments hybridized
7. Those fragments will either show up at locations indicating that they are bigger, smaller, or the same size as the control fragments (normal DNA). INDICATES DELETIONS OR DUPLICATIONS.
36
Describe how Northern Blot is different from Southern Blot:
1. Northern blot uses an mRNA sample instead of DNA
| 2. Detects amount and size of mRNA of interest
37
Describe Western Blot:
1. Western blot uses Protein instead of DNA or mRNA
2. Proteins are denatured with SDS (A negatively charged Detergent) to they will behave like DNA and migrate toward the (+) pole of the gel.
3. They are subjected to gel electrophoresis
4. Gel is transfered to a Nylon Film
5. Add an Antibody that will hybridize to the fragments of interest (an enzyme or substrate)
6. Add a 2nd Antibody Probe that will recognize the 1st antibody and fluoresce.
7. Fragments that fluoresce will indicate smaller, larger, or equal size proteins of interest.
38
Describe Sanger Sequencing:
1. Set up 4 DNA synthesis reactions with:
a. ) ssDNA template
b. ) Radio-labeled Primers
c. ) dNTP's
d. ) DNA Polymerase
2. Add ddNTP's (a different one to each reaction lane
3. When incorporated, these ddNTP's will stop polymerization because they lack free 3'-OH groups.
4. Each reaction is separated by gel electrophoresis and the gel is exposed to X-rays.
5. Labels on the millions of reactions will show the sequence of base pairs.
39
Describe RFLP Analysis:
***RFLP = A single base polymorphism that affects a restriction site***
***Must KNOW what restriction site has been changed before doing the analysis***
Ex: Sickle Cell Anemia: Beta Globin mutation destroys a restriction site (for Dde1)
1. Patient DNA is digested with THAT specific restriction enzyme (Dde1) to isolate the target sequence.
2. Gel electrophoresis of fragments, then transfer to a membrane/film.
3. Add a radioactive probe that is designed to hybridize to that entire portion of the beta globin gene.
4. Expose the film to X-rays, visible fragments will either be longer (restriction site was destroyed) or shorter (restriction site was added).
40
Describe diagnosis of Maple Syrup Urine Disease via RFLP Analysis:
1. Y393N destroys a restriction site (for Sca1)
2. PCR Amplify a 186bp fragment of the disease
3. Digest it with the restriction endonuclease (Sca1) and subject it to gel electrophoresis
4. The single nucleotide change will have produced a longer sequence that will be visible upon UV light exposure.
41
Explain how ASO Probes work:
1. Isolate DNA from white blood cells of patient, amplify, denature, and apply to a gel (2 lanes that each have the same patients DNA)
2. Add a probe complementary to the normal gene in lane 1, and a probe complementary to the mutant gene in lane 2
3. Observe if probes hybridized in each lane to give the genotype of the patient.
* **Only useful if the mutation is KNOWN***
42
Explain Allele Specific PCR:
* **Used to detect Single Nucleotide Changes***
1. Perform PCR, but add primers designed to hybridize to the sequence containing the mutation.
2. Perform 2 tests in which one test is an internal control (the primer is designed to hybridize to the normal allele) and the other test has a primer for the mutant allele.
* **MUST know the mutation sequence***
43
How is RFLP Analysis used with Western Blot?
RFLP Analysis will show IF the polymorphism has occurred (if a restriction site was altered), and Western Blot shows if Protein Expression was affected.
44
List the 4 major intracellular systems affected by cell injury:
1. Membrane Integrity
2. ATP Synthesis
3. Protein Synthesis
4. Chromosome Integrity
45
What is the earliest histological evidence of cell injury?
1. Cloudy swelling
| 2. Lighter staining as a result
46
What is the 2nd sign of cell injury?
1. Hydropic Degeneration: MORE swelling
| 2. Vacuoles Appear: Fluid-filled pockets in cytoplasm, basophils C stain won't show as much (less blue)
47
What is the 3rd early sign of cell injury?
1. Steatosis: Fatty change, accumulation of triglycerides in the cytoplasm
2. Most common in liver --> Caused by alcohol or toxins. Occasionally seen in skeletal and cardiac muscle.
48
List the 3 cellular adaptations to non lethal injury:
1. Atrophy/Hypertrophy: Change in size
2. Hyperplasia: Change in number
3. Metaplasia: Change in differentiation
49
Hyperplasia is the result of _______ while hypertrophy is the result of _______.
1. Increased Cell Division/Hormone Stimulation
| 2. Increased Protein Synthesis
50
How does the body compensate for hypertrophy?
Protein degradation/Autophagy
51
3 of each Tissues commonly affected by Hypertrophy and Hyperplasia Respectively:
1. -->Hypertrophy:
a. ) Cardiac Muscle
b. ) Skeletal Muscle
c. ) Uterus
2. -->Hyperplasia:
a. ) Endometrium
b. ) Red Blood Cells
c. ) Prostate Gland
52
What is a defining characteristic of hyperplasia in affected tissues?
Larger ducts due to increased glandular epithelium
53
3 Tissues commonly affected by Atrophy:
1. Testis
2. Brain
3. Skeletal Muscle
54
Give 2 examples of causes of Metaplasia:
| Is it reversible?
1. Cigarette Smoke: Changes airway epithelium from Pseudostratified Epithelium to Stratified Squamous Epithelium (?)
2. Acid Reflux: Changes esophagus epithelium from Stratified Squamous epithelium to Simple Columnar
3. YES
55
Define Dysplasia and give 3 characteristics:
1. Failure to differentiate (remaining a stem cell)
a. ) Structurally Abnormal
b. ) High nucleus-cytoplasm ratio
c. ) DARK staining chromatin in large nucleus
56
2 Tissues commonly affected by Dysplasia:
1. Cervix
| 2. Skin
57
What danger arises from dysplasia?
Risk of neoplastic formation, due to rapid multiplication of stem cells
58
How will dysplasia be recognizable on a histology slide?
Example: Layers of the skin should show changing epithelium type. Instead most will look the same because they are all still stem cells. (With carcinoma ALL could look the same)
59
Define Neoplasia and compare the 2 types:
1. Cell proliferation in the absence of external stimulation
2. a.) Benign: Slow, localized growth
b. ) Malignant: Fast, invasive growth
60
Describe Linkage Analysis Requirements:
* **INDIRECT detection of mutations***
1. Used for genes known to be linked to a specific marker locus, and it looks for that locus (as a marker of the mutation)
2. Must be used for a large family with several affected people
3. After observing affected AND unaffected individuals markers near their mutations, you can predict which family members have the highest chance of inheriting the marker and, therefore, most likely the mutation.
61
What is Linkage Analysis based on (general concept)?
Recombination during Meiosis
62
What are "Linked Loci"?
Loci that have LOW recombination frequency
63
What are the 2 biggest challenges of using Linkage Analysis:
1. The occurrence of recombination events
| 2. Locus Heterogeneity
64
What is an LOD Score?
Logarithm Of the Odds: Data from multiple families is collected and used to determine a more accurate chance of an individual inheriting a mutation.
65
How can Direct Methods of detection be separated into two types? Give all the examples of each type:
1. Methods that detect ONLY what is being asked for:
a. ) Sanger Sequencing
b. ) Northern/Western Blot
c. ) FISH
d. ) ASO Blot
e. ) Southern-RFLP
f. ) PCR-RFLP
g. ) PCR Sizing
2. Methods able to query the entire genome:
a. ) SNP Chip
b. ) Spectral Karyotype
c. ) Next Generation Sequencing
d. ) Array CGH
e. ) Karyotype/G-Banding
f. ) Expression Arrays
66
PCR product size may be useful in diagnosing ______.
| Give an Example:
1. Triplet Repeat Expansion Disorders BEFORE the symptoms present themselves.
2. Huntington's Disease: PCR fragments will indicate size when UV light is shined on agarose gel. Fragment with 50 repeats or more indicate late onset, and fragments with 100 repeats or more indicate early onset.
67
Describe Cell treatment in prep for G-banding or FISH:
1. Isolate cells from blood sample and grow them in phytohemagglutin.
2. At metaphase, arrest cells with Colchicine/Colcemide.
3. Add hypotonic solution to swell them so they burst onto microscope slide.
4. Fix chromosome and prepare for staining.
68
How does Giemsa Staining work?
Giemsa stain will bind to A-T rich regions, so they will be dark, while the G-C rich regions will be lighter staining
69
How is FISH different than G-Banding?
Uses millions of probes that are only 10-100kb each for higher resolution
70
Differentiate between the two types of FISH:
1. Simple FISH: Probes only hybridize to a specific locus on a chromosome. Search for microdeletions, translocations, AND duplications.
2. SKY Fish (Spectral Karyotyping): Probes hybridize to entire chromosomes, painting each one a different color. Search for complex chromosomal rearrangements, only identifying which chromosome has a mutation.
71
What is unique to FISH that is not true of G-Banding?
FISH can be used at Interphase, not just Metaphase. At this point, it can reveal aneuploidy (trisomy or monosomy)
72
SKY FISH is able to detect _________, because each chromosomes material is indicated by a different color.
BALANCED Translocations
73
How are probes made for SKY FISH?
1. Sequences unique to each chromosome are created in individual tubes.
2. Fluorescently labeled probes (with different colors) unique to each sequence are added to respective tubes.
3. Probes are hybridized to the DNA at metaphase
4. Karyotype may be analyzed to visualize each chromosome in a different color.
74
How is Microarray CGH different than Karyotyping?
It is comparative in that it uses TWO differently labeled DNA sequences to hybridize to a slide, where one is the normal sequence and the other is a patient's potentially mutated sequence. The mutation will indicate COPY NUMBER VARIANTS, so either duplication or deletion.
It CANNOT detect balanced rearrangements.
75
Describe how Microarray CGH is prepared:
1. Sequential DNA fragments representing the entire genome in 1 Mb chromosomal locations are isolated.
2. Millions of probes for EACH 1 Mb fragment are "spotted' onto a slide and immobilized (differently labeled for control vs patient DNA).
3. In each spot, add equal amounts of patient DNA and control DNA.
4. Each spot will indicate if patient DNA has hybridized MORE/LESS than the control to show copy number variants and their locus of origin.
76
How can the design of Microarray CGH be altered?
It can be made to give higher resolution by making exponentially more sequential fragments of smaller size (even 1000bp fragments) to detect deletions/duplications of very small size.
77
How is an Array CGH test read?
X-Axis: Position of sequence on chromosome
| Y-Axis: Ratio of gene copies compared to normal (PLUS indicates duplication, MINUS indicates deletion)
78
Array CGH is the first tier test for _______.
Children with mental impairment or developmental delay
79
What 3 things CAN'T Array CGH detect?
1. Triplet Repeat Disorders (too small)
2. SNP's (way too small)
3. Balanced Translocation
80
Explain what the Philadelphia Chromosome is an example of and how it should and should not be detected:
1. Balanced Translocation between Chromosome 9 and Chromosome 22.
2. Can't be detected be array CGH because it's balanced, but CAN be detected by SKY FISH because it will illuminate the translocated portions as different colors.
81
What gene mutation does the Philadelphia chromosome translocation give rise to? What does it cause?
BCR-ABL Oncogene: Causes Chronic Myelogenous Leukemia
82
What is the main limitation to Array CGH (besides the inability to detect balanced translocation):
Doesn't have a High Dynamic Range: It cannot detect amplifications above a certain number.
i.e. It reads 8 or 10 extra copies the same as 50 extra copies (only indicates a duplication has occurred)
83
FISH can detect insertions/deletions at the ______ level, but can also detect them at the ______ level if using SKY FISH.
FISH = 1 Mb
| SKY FISH = Entire Genome (chromosomal level)
84
Besides Array CGH, what 2 other methods can detect micro-deletions/insertions?
1. Expression Array
| 2. SNP Chips
85
Describe how SNP Chip works:
1. Isolate patient DNA, denature, separate into fragments.
2. Fluorescently label the ssDNA from the patient.
3. Add this to chips containing probes for various alleles for the gene (normal and several common mutations).
4. The SNP array is then scanned by a laser-microscope to see which spots the patient's DNA has hybridized to.
86
What is the main advantage of SNP Chip arrays?
They can detect thousands of SNP's in a single experiment
87
How is each locus on a SNP Chip array read?
One Dot = Homozygous for the SNP
| Two Dots = Heterozygous for the SNP
88
Microarray Analyses like SNP Chip are the first tier test for ______.
Children born with birth defects
89
Describe how cDNA Microarrays work:
1. Isolate RNA from control and patient tissues
2. Create labeled cDNA form the RNA of each sample (via cloning)
3. The two cDNA's created are added to a microarray and will compete to hybridize to the array.
4. Spots on the array will indicate which cDNA there is MORE OF hybridizing to that location, so which tissue is expressing more RNA and therefore more protein.
RED = More Mutant (cancer) tissue cDNA is present
GREEN = More Normal tissue cDNA is present
YELLOW = Equal amount of cDNA are present
90
What is cDNA microarray used for?
1. Comparing the set of mRNA that is expressed in a certain tissue type between cancerous and normal cells.
2. May be used to identify WHICH GENES are or aren't being expressed in a patient.
3. May also be used to compare gene expression between different tissue types.
91
How does Next Generation Sequencing have "greater read depth"?
If multiple successive run of sequencing detect a different base at a certain location, we can continue to sequence it hundreds of times to determine if the sequence is accurate (Showing about 50/50 presence of each base recorded to be at a certain location indicates either a:
- Polyorphism
- Mutation
- Rare Variant
92
Describe the 3 types of substitution mutations:
1. Silent: Nothing changes
2. Missense: Amino Acid is changed
3. Nonsense: Amino Acid becomes a STOP CODON
93
What are the two types of Transition mutations? Give an example of one:
1. Purine to Purine: (A->G or G->A)
2. Pyrimidine to Pyrimidine: (T->C or C->T)
Ex: Hemochromatosis is a G->A mutation that changes the normal HFE gene into the C282Y mutation
94
What is a Transversion mutation? Give an example:
1. Purine to Pyrimidine (A->T or G->C)
2. Pyrimidine to Purine (T->A or C->G)
Ex: Sickle Cell Anemia is an A->T mutation that changes the normal HbA gene to the HbS mutation
95
Isozymes:
Enzymes that have different amino acid composition but perform the same action
96
What are the 3 main enzymes we use as markers of cell injury?
1. Creatine Kinase
2. Lactate Dehydrogenase
3. ALT:AST Ratio
97
How many subunits do Creatine Kinase and Lactate Dehydrogenase have respectively and how are they denoted?
1. Creatine Kinase: 2 Subunits (M and B)
| 2. Lactate Dehydrogenase: 4 Subunits (2H and 2M)
98
Where is Creatine Kinase found and what reaction does it catalyze?
1. Cytosol and Mitochondria
| 2. Conversion of Creatine into Creatine Phosphate (CrP) via ATP hydrolysis
99
Most Creatine Kinase is in the isozyme form of _______. Where is the rest of the body's Creatine Kinase and why is that important?
1. CK-MM = 98% in skeletal muscle
| 2. CK-MB = 2% MOSTLY in Heart Muscle, used as a marker for MI
100
How is LDH used as a marker?
1. HIGH LDH1:LDH2 ratios indicate possible MI
| 2. HIGH LDH5 indicates hepatic or sk. musc. damage
101
Where is LDH2 normally found to be high and low?
1. HIGH = RBC's
2. LOW = Heart
So a blood sample would indicate a LOW LDH1:LDH2 ratio for a normal healthy individual
102
What other enzymes will be seen as markers of an MI?
Cardiac Troponin "I" and "T"
| cTnI and cTnT
103
After an MI, the total serum concentration of Creatine Kinase contains mostly _________. But what part of the serum CK levels is the important indicator of MI?
1. CK-MM
2. If the CK-MB level is more than 6% of the total OR if CK-MB is more than 3% AND Troponins are increased, then it is significantly increased and indicating MI possibility.
104
How are biomarkers detected in the serum?
ELISA: Enzyme-linked Immunosorbent Assay
| also Electrophoresis and Radioimmunoassay
105
Why is Troponin C not measured as a biomarker?
Because it doesn't have an isoprotein specific to the heart (there is no cTnC)
106
What biomarkers should be detected for 0-6 hours, 12 hours, and 2 days after the onset of symptoms respectively?
- ---->0-6 Hours:
a. )Myoglobin
b. )CK-MB
c. )HFABP (Heart-F.A.-Binding Protein)
- ---->12 Hours:
a. )CK-MB
b. )Total CK
c. )cTnI and cTnT
- ---->2 Days:
a. )cTnI and cTnT
b. )LDH isozymes/ratio
107
When do Myoglobin, CK-MB, and Cardiac Troponins peak respectively?
1. Myoglobin: 6 hours
2. CK-MB: 1 day
3. cTnI/cTnT: 1-2 days (still measurable up to 6 days later)
108
What 3 measurements indicate decreased liver function vs what 2 measurement indicate decreased liver integrity?
1. Function Decreased:
a. ) HIGH Ammonium
b. ) HIGH Bilirubin
c. ) LOW Albumin
2. Integrity Decreased:
a. ) ALT:AST Ratio
109
Liver Damage NOT related to alcohol abuse =
| Liver Damage FROM alcohol abuse =
1. HIGH ALT:AST
| 2. HIGH AST:ALT > 2
110
How is biliary excretory function measured?
| What 2 things might high levels of these indicate?
1. ALP-2 (Alkaline Phosphatase in bile canaliculi of pancreas and liver)
2. GGT (Gamma-Glutamyl Transpeptidase)
Might indicate:
a.) Gallstones
b.) Pancreatic Tumor
111
What does GGT do and how does this relate to its measurement?
1. It cleaves Glutathione to form Amino Acids
| 2. If it's function is decreased, this is indicated by INCREASED Glutathione (leads to liver and gallbladder problems)
112
What 2 things cause acute pancreatitis vs what 3 things cause chronic pancreatitis? Give the serum levels that indicate acute vs chronic as well:
-----> Acute:
1.) Alcohol Abuse
2. Gallstones
Serum: HIGH amylase and lipase
-----> Chronic:
1. CF
2. Hypertriacylglycerolemia
3. HEAVY Alcohol Abuse
Serum: Lipase:Amylase Ratio > 2 indicates that damage is characteristic of alcohol abuse
113
What is ALP-2 involved with and what might high levels of it in the plasma indicate?
1. It cleaves PO4- groups in bone to create an alkaline pH for Ca2+ deposit onto collagen of bone.
2. Increased levels = Bone Disease such as Paget's Disease or tumors.
* **HIGH levels are NORMAL in women during pregnancy or children with growing bones***
114
3 Reasons for Increased serum ALP:
1. Pregnancy/Child Development
2. Bile Duct Obstruction (****ALP-1****)
3. Bone Disease (****ALP-2****)
115
3 Reasons for GGT Increased in serum:
1. Gallstones
2. Alcohol Abuse
3. Certain drugs
116
What is the donor of BOTH sugar and phosphate for BOTH purines and pyrimidines?
PRPP
117
What amino acids are the building blocks of purine de novo biosynthesis? (4)
What initial compound do they form?
1. Glycine
2. Aspartate
3. Glutamine
4. THF
They initially form AMP and GMP
118
What molecules are formed from purines following cell death or nucleotide turnover? What two pathways can they enter and what will they become next?
1. Adenine, Guanine, Hypoxanthine
2. Cell Recycling-->AMP/GMP
3. Degradation-->Uric Acid
119
What molecule is the donor of Carbon for BOTH purine and pyrimidine de novo biosynthesis?
THF: The folate form of Uric Acid
| and CO2 donates some carbon as well
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How is Ribose Phosphate derived?
From the Pentose Phosphate Pathway
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What molecules are used in the initial step of Pyrimidine de novo synthesis of UTP and CTP?
1. Aspartate
2. Glutamine
3. CO2
* **NOT Glycine or THF--> Only Thymidine uses THF out of all the pyrimidines***
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How is de novo synthesis of pyrimidines different from purines?
FIRST the C and N atoms are donated to form the pyrimidine ring, THEN the PRPP is used for adding the PO4- and Sugar group.
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Amplification:
Enzymes activating other enzymes
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Integration:
When 2 signals have opposite effects that result in a net response
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Gated-Ion channel examples: (3)
1. Ach
2. GABA
3. Glycine
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Receptor Enzyme Example:
Insulin
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4 Examples of 2nd Messengers:
1. cAMP
2. cGMP
3. IP3/DAG/Ca2+
4. Nitric Oxide (NO)
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Which receptor does the Glucagon receptor behave most like?
The Beta Adrenergic receptors
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How is cAMP activity stopped?
| What two drugs prevent this inhibition of cAMP activity?
1. Phosphodiesterase hydrolyzes it to 5'-AMP
| 2. Caffeine and Theophylline
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Give an example of covalent modification of an enzyme causing its inhibition:
ADP-Ribosylation of G-alpha subunits results in their continued stimulation (inhibits GTPase activity so they can't turn off)
Ex: Cholera Toxin: Continuously active G sub S
Pertussis Toxin: Continuously active G sub I
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How is the CFTR regulated by covalent modification?
Cholera toxin inhibits the G alpha sub S GTPase activity, so it continuously stimulates Cl- release from the cell.
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Function of cGMP:
2nd messenger for Smooth Muscle relaxation, platelet aggregation, and the visual system
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How is cGMP formed/activated? How is it different than normal GPCR pathways?
1. By Guanylate Cyclase
2. It ISN'T a GPCR, because Guanylate Cyclase is an integral part of the receptor that isn't associated with G-proteins. It activates PKG
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Function of Nitric Oxide (NO):
Activation of Guanylate Cyclase in vascular smooth muscle to cause relaxation by decreasing phosphorylation of myosin light chains
