Exam 2 Flashcards
1
Q
Epigenetics (def)
A
DNA expression w/o change in DNA structure caused by external/environmental factors
-can be heritable
-allows for differing cell types (w/ same DNA)
2
Q
Diethylstilbestrol
A
Potent estrogen drug
Given for miscarriage prevention
= modified child epigenome & reproductive abnormalities
3
Q
DNA methylation
A
gene silencing
methyl groups added to histone tails
4
Q
DNA methylation process
A
CpG methylation (cytosine-phosphate-guanine)
Methyltransferase adds methyl group = 5-methylcytosine
RNA polymerase cannot bind = gene silencing
HDACs recruited –> chromatin condenses! (heterchromatin)
5
Q
Histone modifications
A
Methylation
Acetylation
Phosphorylation
6
Q
Histone tail structure
A
amino acids | positively charged
Modified histones –> neutralized charge
7
Q
Histone Methylation
A
Increase or decrease transcription (depend on number and what is methylated- multiple histone tails can be methylated)
-Arginine (R) & Lysine (K)
8
Q
Methylation Process
A
histone methyltransferase adds methyl groups
= increase or decrease gene transcription
9
Q
Acetylation
A
promotes transcription
neutralizes DNA charge
loosens DNA (euchromatin)
10
Q
Acetylation Process
A
HATs (histone acetyltransferases) remove methyl and adds acetyl groups = change proteins produced
11
Q
Phosphorylation
A
decrease transcription (condense DNA=heterchromatin)
Can be phosphorylated- Serine | Threonine | Tyrosine
12
Q
Phosphorylation Process
A
phosphate groups added by kinases/phosphatases
Aurora B: condenses chromatin (heterochromatin)
Protein kinase: de-condense (euchromatin)
13
Q
What epigenetics is cancer associated with?
A
unmethylated cytosines | acetylated histones
High oncogene expression
deacetylated histones | methylated cytosines
Low tumor suppressor genes
14
Q
Oncogene (def)
A
overactive/expressed mutated gene form
cancer accelerator
15
Q
Proto-oncogenes
A
normal gene involved with cell growth
16
Q
Oncogene example
A
Ras point mutation
17
Q
Tumor Suppressor Genes (def)
A
slow cell growth | assist in DNA repair
cancer decelerator
18
Q
Tumor Suppressor Gene Example (which leads to cancer)
A
P53 protein stops cell cycle
cells replicate w/ mutated DNA = cancer
Caused by heat, radiation, chemicals
19
Q
Tumor Cell Lines (def)
A
Tumor cells will grow in petri dish, unlike normal cell which die
20
Q
HDAC inhibitors
A
cancer treatment
drug which kills cancer cell lines
effect in Mast Cell Tumor
21
Q
RNAi (def)
A
RNA interference
inhibits specific mRNA which decreases protein expression
Types: siRNA | shRNA | miRNA
22
Q
siRNA (def)
A
small interference RNA
silences protein
23
Q
siRNA structure
A
double-stranded | 20-25 nucleotides | exogenous | degrades quickly
24
Q
siRNA process
A
AGO protein complex peels passenger (sense) strand –> becomes RISC
RISC breaks up mRNA = silencing
in cytosol
25
What RNAi silences FVR (feline herpesvirus 1) & how?
siRNA
targets viral protein D required for FVR replication = silencing
26
shRNA (def)
short hairpin RNA
central section of RNA "folds over"
27
shRNA structure
1 stranded and folds to 2 stranded RNA
exogenous | long-lasting
28
shRNA process
Dicer chops "hairpin" part = silence mRNA
constantly reproduced so long-lasting
*in cytosol*
29
Ago
Argonaut
Protein complex used in silencing process of siRNA to peel off passenger (sense) strand
30
RISC
RNA-induced silencing complex
breaks up mRNA for silencing in siRNA
31
Explain shRNA in cattle / texel sheep
Myostatin inhibits muscle growth in cattle
Mutation of myostatin (shRNA) = big muscles!
32
miRNA (def)
micro-RNA
targets several mRNAs for silencing
33
miRNA structure
target several mRNAs
endogenous | long RNA precursors (pri-miRNA)
34
miRNA process
made in nucleus --> transported to cytosol
pri-miRNA --> pre-miRNA by Drosha
Dicer chops off hairpin
mRNA degraded either by cleaving or suppressing during translation = silencing
35
Common diseases with miRNA
cancer
neurodegenerative
cardiovascular
viral infections
36
Explain miRNA's role in Canine Mammary Cancer
miRNA-21 decreases tumor suppressors
miRNA-15a decreases oncogenes
=cancer
37
Pros/Cons of using RNAi for therapeutic purposes
Pros: more specificity than chemical drugs
Cons: price | delivery
38
Antagomirs
inhibit miRNAs that are causing issues (ie cancer)
39
3 Types of Antagomirs
Inhibitors
Sponges
Masks
40
Inhibitor (Antagomir type)
floods cells with passenger strands to take up the guide strands so miRNA cannot bind
41
Sponges (Antagomir type)
synthetic RNA used as "decoy" to soak up complexes (miRNA cannot cause effect)
42
Masks (Antagomir type)
Bind to target sequence on mRNA but w/o Arg
Act like "receptor-blocker" so miRNA cannot bind
43
Gene Therapy (def)
introduces new genes to restore or add gene expression
44
What disease is a good candidate for gene therapy and why?
Neuronal Ceroid Lipofuscinosis
only single nucleotide deletion
45
Forms/Modes of Gene Therapy
Direct
Cell-Based
46
Direct (form of gene therapy)
Inject DNA that encodes for a protein
47
Cell-Based aka indirect (form of gene therapy)
inject DNA into petri dish cells, then patient
use cells from same patient to prevent immune response
48
Types of Gene Therapy
Somatic gene therapy
Germline gene therapy
49
Somatic Gene Therapy
DNA transferred to patient only
no effect on progeny
50
Germline Gene Therapy
DNA transferred to eggs/sperm
passed to progeny (permanent)
51
Gene Therapy Process
Cut & open plasmid
insert DNA and make protein
52
3 considerations for effectiveness of gene therapy
Packing/delivery to cell
Sufficient protein expression
Tissue specificity
53
Delivery Methods for Gene Therapy
Direct injection
Viruses
Nanoparticles
54
Viruses (for gene delivery) types
Adenovirus
Adeno-associated virus
Lentivirus
55
Why are viruses used for gene delivery in gene therapy?
can be produced for a lifetime if integrated into host DNA (via reverse transcriptase)
56
Pros/Cons Adenovirus in gene therapy delivery
Pros: good at getting into cell
Cons: short efficacy | immune response | no cell specificity
57
Pros/Cons Adeno-associated viruses in gene therapy delivery
Pros: long-lasting | cell specificity
Cons: small | limited gene delivery | possible mutation
58
Pros/Cons Lentiviruses for gene therapy delivery
Pros: last lifetime | cell-specificity | low immune response
Cons: could interrupt functional gene | from HIV
59
Nanoparticles (gene delivery type)
synthetic particles (lipids, polymers, metals) that encase DNA for delivery
60
Pros/Cons of Nanoparticles for gene delivery
Pros: easy to make | no risk of infection | low immune response | no size limit
Cons: not efficient in transport | no cell specificity
61
2 ways to achieve tissue specificity
tissue-specific promoter
directed delivery to target cells
peptides engineered on surface only
recognized by certain cell types
62
Totipotent stem cell
can make any cell
63
Pluripotent stem cell
can make any cell except zygotes
64
Multipotent stem cell
can make several cell types within lineage (ex: multiple WBC types)
65
Unipotent stem cell
can only make one cell type
66
Embryonic stem cells
pluripotent (generate many cell types)
formed from "inner mass"
embryo gets destroyed
can lead to uncontrolled tumor/growth
67
Adult stem cells
multipotent or unipotent
derived from patient (no immune response)
less chance for tumor growth
68
iPCS
induced pluripotent cells
normal cell made into pluripotent stem cells
no immune rejection
over-expresses oncogenes (possible cancer)
69
Allogeneic stem cells
from different individual but same species
70
Syngeneic stem cells
from different identical individual (twin)
71
Autologous stem cells
from same individual
72
Genetic Engineering (def)
exogenous DNA or mod of genome that transmits to progeny
73
Types of Genetic Engineering
Transgenic
Knockout
Knock-in
74
Transgenic (genetic engineering)
foreign DNA added to genome
75
Transgenic gene process
-Make transgene (generic or cell specific promoter)
-Inject into egg (male pronucleus of fertilized ovum)
-Insert transgene into genome (random)
76
Transgenic pros/cons
Pros: easy | quick | fine-tuning
Cons: random insertion so possible mutation | overexpression
77
Knock-out (genetic engineering)
suppress/delete endogenous gene
gene cannot express
78
Knock-out Process
-make targeting construct
-introduce embryonic stem cells
-integrate into genome
-inject ES cells into blastocysts for implanting into pseudo-preg female
79
Knock-in (genetic engineering)
mutations added into endogenous gene
80
CRISPR/Cas9 technology
gene-editing in embryonic stem cells
single guide RNA directs nuclease into specific gene site which activates gene and cuts out DNA
81
Knock-in Process
swap normal piece DNA with mutation
82
Knock-in & Knock-out pros/cons
Pros: no random insertion | fine-tuning
Cons: long | expensive | embryonic lethality | protein w/ false genotype possible
83
Purposes of Genetic Engineering
research
food production
therapeutics
disease prevention
84
Why are mice used for genetic engineering?
well-defined genetics
easy to isolate and maintain ES cells & ova
short gestation period
85
Knock-out Pigs
generated virus-resistant pigs (PRRSV disease)
Knocked out SIGLEC1 gene for virus binding
86
Transgenic Salmon
express growth hormone transgene
larger growth in short time
possible problem with wild release
87
Transgenic Goats
antithrombin used for human clotting disease
transgenic goats produce antithrombin
promoter switched on in mammary tissue
secreted in goat milk
88
Transgenic Cattle
Lysostaphin is protein that protects cow from mastitis
cows = mastitis-resistant
89
Rabbit Hemorrhagic Disease (symptoms & cause)
Viral disease (RHDV2-rabbit hemorrhagic disease virus-2)
lethargy | decreased appetite | hypothermia | down platelets | heart issues | protein/heme in UA
Death in 24 hours
Necropsy: discolored liver | hemorrhage | pericardial effusion
90
Virus characteristics
MUST have host for replication
Acellular (NOT cells/organisms)
Replicate by assembly
91
Latent virus
viral genome in host but inactive (not replicating)
92
Productive virus
active viral genome replication
93
Capsid (def & function)
protein coat of viruses
function: protect genome
94
Capsid shapes
icosahedral or helical
95
Viral envelope
membrane layer
Not in all viruses
made from host cell's plasma membrane (so it can evade immune system)
96
Types of viral structures & example viruses
Naked Icosahedral (adeno)
Naked Helical (tobacco mosaic-non animal)
Enveloped Icosahedral (lentivirus, flavivirus)
Enveloped Helical (rhadbo, corona)
Complex (poxviruses, bacteriophage)
97
Viral genome
RNA or DNA
required for viral life cycle and making new viruses
relies on host cell for translation, metabolism, membrane synthesis
98
Monopartite genome
linear genome of virus
99
Segmented genome
multi-molecular parts of viral genome
reassortment & variants possible
100
Parts of a Virus
Capsid
Envelope
Genome
Protein
101
Nonstructural protein
protein "inside" portion of virus
produced early-on
importance: genome
102
Structural protein
protein "outside" portion of virus
produced later on
importance: structure (capsid)
103
Steps of Virus Life Cycle
Attachment
Entry
Uncoating
Replication
Transcription
Translation
Assembly
Release
104
Attachment phase of virus
virus binds to host cell receptors
mediated by glycoproteins (on capsid/envelope)
105
Entry phase of virus
virus enters host
2 entry mechanisms:
Endocytosis (no capsid)
Membrane fusion (w capsid) viral contents into
cell, capsid fuses at membrane surface
106
Uncoating phase of virus
capsid removed
necessary for viral replication
107
Replication phase of virus (DNA/RNA virus)
via assembly
DNA viruses- in nucleus | own DNA polymerase or host cells
RNA viruses- in cytosol | own RNA polymerase
108
Replication process of virus exceptions
Retroviruses (cDNA in cytosol | "intermediate" DNA in nucleus)
Poxviruses (DNA replication in cytosol via factories)
109
Transcription phase of virus
must produce sense mRNA (+mRNA) for host cell machinery to translate all viruses
110
Translation phase of virus
fully dependent on host cell
makes single polypeptide that's cleaved
111
Assembly phase of virus
occurs at replication site (nucleus or cytosol)
self-assembly
112
Types of viral assembly
spontaneous (simple)
directed (complex)
113
Release phase of virus types
Lytic --> kills cell
Non-lytic --> does not kill cell
114
Lytic release
non-enveloped viruses rupture plasma membrane and kill cell
*fast process*
115
Non-lytic release
enveloped viruses "bud off" membrane (no killing of cell)
*slow process*
116
Antiviral therapies
Vaccines
Drugs
117
Vaccine as antiviral therapy types & examples
inactivated virus (rabies, WNV)
live, attenuated virus (canine adenovirus)
DNA plasmids (WNV, feline leuk)
mRNA (SARS-CoV2, zika)
recombinant capsid proteins (hepatitis, papilloma)
118
Drugs as antiviral therapy & example
target viral DNA replication
example: acyclovir | tenofovir (inhibit reverse transcriptase)
119
4 Classifications of Parasites
-Arthropods (diptera-flies, mosq; mites, ticks)
-Protozoa (apicomplexans-cytaux & crypto; flagellates)
-Platyhelminths (cestodes-tapeworm; trematode-fluke)
-Nematodes (strongylida-hooks, rhabditia, spiruids)
120
Cryptosporidium
protozoan parasite (Cryptosporidium parvum)
affects all mammals + humans!
zoonotic disease
121
Crypto symptoms/transmission
S: rapid/acute D+ | long asymptomatic period
T: feces | waterborne
122
Cryptosporidium (life cycle)
Oocyst encases sporozoites
Embed in GI lining
Transmitted via oocysts in feces
123
Oocyst
hard shell casing | infectious agent of parasites
124
Cryptosporidium (cell uptake)
Micronemes & Rhoptries move to cell surface & interact with epithelial host cell surface
Get into cell via actin remodeling
Feeder organelle transfers nutrients from host
125
Micronemes & Rhoptries
receptor-like proteins in Crypto that interact with host cell epithelial membrane
Induce actin remodeling for cell uptake
126
Actin Remodeling
host cell actin forms stress fibers which bring parasite into host cell
127
Feeder organelle
allows for nutrient transfer from host to parasite via cpABC transporters (in crypto)
128
cpABC transporters
part of host cell that takes in nucleotides from parasite which allows for its replication
allows for feeder organelle to form (for parasite nutrition from host)
129
Cryptosporidium D+ process
parasite causes decrease in absorption (microvilli) of intestines
Intestines retain and add water
130
Treatment for Cryptosporidium
none specifically approved
Current: nitazoxanide (antiprotozoal in cattle)
Potential: lectins- block parasite anchoring
cpABC inhibitors- starve parasite
131
Cytauxzoon (symptoms & transmission)
Apicomplexan protozoan (Cytauxzoon felis)
ONLY affects cats
S: fever | anemia | icterus | enlarged organs | high mortality (60%)
132
Cytauxzoon transmission
T: lone star tick (amblyomma americanum)
133
Cytauxzoon disease mechanisms
Initial stage: macrophage infection = clog vessels
Progression: RBC infected = transmission by tick blood meal
134
Cytaux life cycle
-Sporozoites --> enlarged macrophage
-Enter RBC --> transmission (tick blood meal)
135
Cytaux cell uptake
"Zippering" on host cell
Enter via phagocytosis (evade immune system)
Proteins released into cell for replication
136
Cytauxzoon Treatment
Only tick prevention
137
Prion Disease
Zoonotic disease caused by misfolded prion protein
PrPc (normal) --> PrPsc (abnormal)
138
Prion Disease examples
Scrapie (sheep/goat)
Chronic Wasting Disease (cervids)
Bovine Spongiform (cattle)
TSE (non-human primates)
139
Prion Disease transmission
feed, soil, brains
140
Prion Disease resistant/sensitive to...
R: heat | pressure | UV
S: proteinase K | denaturants (urea)
141
Prion Disease uptake
passed to dendritic cells
transferred to enteric nerves (GI)
brought to CNS
142
PrPc
Normal/endogenous prion form
a cell surface protein
143
PrPc structure
3 alpha helices
anchored to plasma membrane by GPI (phospholipid)
144
PrPc function
1. copper regulation (prevents buildup)
2. control of axon/neurite outgrowth (prevents overgrowth)
145
PrPsc structure
B-sheet
Aggregate B-sheet to create insoluble protein fibril
Fibrils build up in brain = prion disease
146
Steps for Prion Disease
1. Conversion (PrPc --> PrPsc)
2. Seeding
3. Propagation
4. Toxicity
147
Types of Prion Conversion
Spontaneous
Nucleation
Template/Assisted
148
Spontaneous prion conversion
PrPc mutates = PrPsc
by genetic mutation (but unfavorable)
149
Nucleation prion conversion
PrPsc binds to PrPc = convert to abnormal
150
Template/Assisted prion conversion
PrPc unfolds and PrPsc mutates it during unfolded state
151
Seeding (prion disease)
PrPsc aggregate into seeds
Create fibrils which build-up in brain
Seeds spread to other neurons
152
Propagation (prion disease)
PrPsc seeds spread to other cells in brain
153
Toxicity (prion disease)
Cell internalizes PrPsc and tries to digest
Unfolded proteins build-up in ER
ER stress = massive Ca2+ into cell --> death
154
Potential Treatment of prion disease
-target prion protein (antibodies reduce PrPc so PrPsc cannot develop)
-inhibit PrPsc toxicity (prevent aggregation or reduce ER stress)
155
Tetanus Toxin
potent bacterial toxin
horses greatly affected
caused by Clostridium tetani which attacks NMJ
156
Clostridium tetani
gram-positive | anaerobic bacteria
causes tetanus toxin
157
Tetanus Toxin forms
Vegetative (infectious | rod-shaped)
Spore (long-lasting)
158
Tetanus Toxin symptoms
stiffness | difficulty swallowing | muscle spasms (lock-jaw)
159
Tetanus Toxin transmission
soil, feces
160
Structure of Tetanus Toxin
Light chain + Heavy chain
161
Light Chain of Tetanus Toxin
Protease | "business end"
162
Heavy Chain of Tetanus Toxin
for neuron binding & transports light chain
163
Infection Process of Tetanus Toxin
1. Transported from Ach neuron (excitatory) --> GABA neuron (inhibitory)
2. Toxin endosome acidified
3. Heavy chain of toxin forms pore
4. Light chain released into cytosol
5. Light chain digests SNARE proteins
6. cleaving of SNARE prevents GABA
7. Constant Ach release = constant contraction
164
SNARE Protein
Ca2+ driven protein required for binding/fusion of vesicles for NT release
165
V-SNARES
proteins on vesicles with NT
required for NT release
166
T-SNARES
proteins on plasma membrane
required for NT release
167
Tetanus Toxin Treatment
Current: vaccine
immediate anti-toxin administration
High-dose antibiotic + clean wound
Potential: antibodies (tetanus-->botox)
lectins
inhibitors of light chain
168
Anthrax Toxin
bacterial toxin
cause: Bacillus anthracis
affects all mammals, mostly ruminants
169
Bacillus anthracis
gram-positive bacteria | causes anthrax toxin
170
Anthrax Toxin Forms
1. Vegetative (rods | fragile)
2. Spore (ovoid | requires O2 | last for years)
171
Routes of Anthrax Infection
Cutaneous- skin abrasions (survivable)
Gastrointestinal- ingest food/soil (fatal)
Oropharyngeal- breathing in
172
Forms of Anthrax
Peracute
Acute
Subacute
173
Peracute
Anthrax form = Sudden death
Mainly ruminants
Symptoms: tremor | neck edema/asphyxiation | bloody discharge
174
Acute
Anthrax form | rapid onset (days)
Symptoms: tremor | neck edema/asphyxiation | bloody discharge
175
Subacute
Anthrax form | chronic (days to weeks)
Symptoms: gradual neck edema | anorexia | V+/D+ | GI necrosis
176
Anthrax Toxicity proteins involved
Protective Agent (PA) - carrier to get factors into cell
Lethal Factor (LF)
Edemic Factor (EF)
177
ANTXR1 & 2
endogenous receptors that bind anthrax toxin
178
Mechanism of Anthrax Toxin
cell brings in toxin
pore in cell by increasing acidity = release EF & LF
EF converts ATP to cAMP = cell "leaky" = edema
LF cleaves MAPKK (survival protein) = cell death
179
MAPKK
survival protein in cell that inhibits apoptosis in normal cell
cleaved by lethal factor in anthrax
180
Anthrax Toxin treatments
Current: antibiotics | vaccine
Potential: anti-PA antibodies | EF inhibitors | "decoy" proteins bind to PA | anthrax receptor antagonists
181
Gossypol
toxic agent in cottonseed
182
What apoptotic pathway is stimulated by gossypol?
intrinsic apoptotic pathway --> cell death
183
Extrinsic Apoptosis Pathway
receptor-mediated
cell folds in on itself
uses caspase for cell death
184
Caspase
enzyme that allows for cell death in extrinsic apoptosis pathway
185
Intrinsic Apoptosis Pathway
use mitochondria in roundabout way to lead to cell death (used by gossypol to make cottonseed toxic)
186
Intrinsic Apoptosis Pathway mechanism
ischemia | UV | hypoxia activate Bax
Mitochondria release CytC
CytC binds to Capase = cell death
non-toxic cell inhibits pathway via Bcl2
187
Bax
hole/pore created in outer mitochondrial membrane created by ischemia, UV, hypoxia in intrinsic apoptotic pathway
188
CytC
used in mitochondria for ETC
released during intrinsic apoptosis pathway = binds to caspase for cell death
189
Bcl2
inhibits intrinsic pathway so no cell death occurs in cell w/o toxin
190
Yew (Taxus)
plant toxin; toxic to all animals, monogastrics especially
191
Yew (Taxus) toxic agent
Taxine-A & Taxine-B
192
Taxine-A & Taxine-B
taxine alkaloids (lots of benzene)
active (no metabolism needed)
cardiotoxic (affect heart)
193
Yew toxicity mechanism
affect AP conduction
block Na+ & Ca2+ channels
PR, QRS, ST phases elevated (on ECG)
= arrythmias (decrease cardiac function)
194
Perilla mint (Perilla frutescens)
invasive plant toxin | affects LA (mostly cattle)
insensitive in pigs/dogs
Need to be metabolized for toxicity
195
Perilla mint toxic agent
Perilla ketone
196
Perilla mint toxicity mechanism
P450 enzymes convert perilla ketone to toxic form
PK activates TRP Ca2+ channel
Ca2+ into cell = contraction of endothelial/epithelial cells
--> pores between cells = fluid in lungs
197
TRP
transient receptor potential channels
NSCC
198
P450 enzymes
convert perilla ketone --> toxic form
199
Pigweed (Amaranthus species)
invasive | toxic in LA | non-toxic in SA
plant toxin
200
Symptoms of pigweed toxin
weakness | ataxia | recumbency | symptoms are a few days | usually fatal
201
Pigweed Toxic Agent
oxalates
202
Pigweed toxicity mechanism
oxalates bind to Ca2+ in blood = crystal formation --> renal failure
203
Gossypol as therapy
anti-cancer agent
cancer increases Bcl2
gossypol decreases Bcl2 = cell death
204
Name 2 plant toxins used as chemotherapeutics
Taxol (paclitaxel)
Vincristine
205
Which plant toxin is used for heart failure? How does it help?
digoxin
increases cardiac contractility
206
Which plant toxin is used as an anti-diabetic agent?
Curcumin
