Open questions 1 Flashcards
Describe the ayoma staining method
The sample is fixed in formalin containing CdCl2
that makes Golig membranes argirophilic
o Then the tissue is impregnated with AgNo3 and
Golgi membranes tend to bind Ag ions
o In the next step, Ag ions are reduced to black metal
Ag by hydrokinone
o After embedding and cutting the sections are
stained with Orange G and Safranine
o Results: dictiosomes of Golgi are black, the
cytoplasma is yellow and nuclei are red
Describe the location, the structure and the main functions
of the nuclear lamina
The nuclear lamina is a scaffold-like network of protein
filaments surrounding the nuclear periphery. Consists
mainly of intermediate filament proteins Lamin A/C and B, which together form a complex meshwork underneath the inner nuclear membrane.
- Functions:
➢ Maintenance of the nuclear envelope and nuclear shape.
➢ Spatial organization of the nuclear pores within the nuclear membrane.
➢ Regulation of transcription.
➢ Anchoring the interphase heterochromatin.
➢ Role in DNA replication.
Describe Feulgens reaction. What are the steps and the
result of it?
What can be detected by this method?
Whatkind of cytological method is it?
DNA can be detected by this way
o This reaction is made in two steps
- In the first HCl is used for a mild acidic hydrolysis à RNA is dissolved from the cells, then purine bases are separated from the DNA,then 2-deoxyriboses turn into aldehyde
- In the second step aldehyde groups form a
colored product with Schiff reagent
(leukofuchsin)
Describe the mechanism(s) by which water molecule pass
the plasma membrane?
What are the similarities and differences between the processes?
- Diffusion and osmosis
- Aquaporins: integral membrane proteins that forms pores, mainly facilitating transport of water between
cells
Similarities: They both don’t require energy (ATP)
Differences: Simple diffusion happens through membrane other uses aquaporins (channel proteins)
Describe the morphological (size) and the chemical
structure of eukaryotic ribosomes. Where are they located
in the cells?
- Large subunit (49 ribosomal proteins + 3 rRNA moleclues) and small subunit (33 ribosomal proteins
+ 1 rRNA molecule) - Located in cytoplasm or on the rER
List the major types of membrane lipids. Where are they
synthesized?
- Phospholipids on rER
- Glycolipids in Golgi (trans?)
- Cholesterol on rER
What does glycosylation mean?
What are the types?
Where does it happen?
What is the significance
of glycosylation?
Glycosylation is the enzyme process that links saccharides to produce
glycols. There are two types of it:
1) N-glycosylation
2) Oglycosylation
- N-Glycosylation is addition of oligosaccharides to the NH2 of ASP
(asparagine) . It starts in the ER and continues in the Golgi.
Oglycosylation is the addition of oligosaccharide to the OH of SER, THR
- It happens in med and trans Golgi
- (Glycosylation is addition of oligosaccharide to molecules e.g.. proteins)
- Main functions: protect a protein from degradation, hold it in the ER until it is properly folded or help guide it to the appropriate organelle by serving as a
transport signal for packaging the protein into appropriate transport vesicles
What are the difference between constitutive and regular
secretion (exocytosis)
o Constitutive: is performed by all cells and serves the release of components of the ECM or delivery of newly synthesized membrane proteins that are incorporated in the plasma membrane after the fusion of the transport vesicles
o Regular secretion: Non-constitutive requires an external signal, a specific sorting signal on the vesicles, a clathrin coat, as well as an increase in
intracellular calcium
List five significant and different features of human
mtDNA (mitochondrial DNA).
o Circular DNA
o No histones
o No chromatin
o Mitochondria are derived only from mitochondria:
mitochondrial DNA is replicated and the mitochondrion divides by cleavage
o All mitochondria of an individual originated from
the mother
Describe shortly autolysis and autophagocytosis. What is
the major difference between them?
o Autophagy: very important. Useful for digesting harmful molecules.
o Autolysis: uncontrolled process where lysosome enzymes get out of control and digest everything
What is the purpose of embedding in the microscopic microtechniques?
Which substances are used for
embedding?
In which cases is embedding not needed?
o Purpose: to provide a mechanical support. The
technique of infiltrating the fixed tissue with paraffin is called embedding
- Alcohol (dehydration)
- Xylene (for removal of alcohol)
- Paraffin (embedding agent)
o Freezing (physical fixation maked the tissue solid enough to section it without embedding
Whic 2 RNA processing stops occur on transcripts destined to become rRNA?
What is the significance of these modifications?
Where do these steps happen in the cell?
1) 5’ end capping (cotranscriptional modification)
Adding methyl guanine cap to the 5’
2) 3’ end tailing (co- transcriptional modification) Adding poly A tail to 3’
3) Splicing (co-transcriptional modification) by splicosome (snRNPs) cutting out the introns and binding exons. The process regulate and stabilise mRNA synthesis.
Happens in the nucleus.
Where are the mitochondrial proteins synthesized?
How are they targeted and transported to their destination?
Mitochondrial proteins are synthesized in cytosol on free ribosomes
The proteins synthesized on free ribosomes have mitochondrial
localisation signal on there N-terminus.
This binds to the outer membrane receptor (this receptor can recognize the mitochondrion localising signal).
The 2 membranes (outer and inner) are temporarily linked forming a membrane contact site. These sites are the translocon molecules TIM and TOM, which form the tunnel for proteins to enter mitochondria.
TOM is in action to enter for outer membrane protein and TIM is in action for inner membrane protein
List the main steps of the microscopic microtechniques.
Give the name of the substances or instruments that is commonly used in a given step.
- Sample
- Fixation: to perserve tissue as life-like as possible.
• Physical
• Chemical – enzymes are denatured irreversibly.- Aldehydes (formaldehyde)
- Heavy metal ions
- Alcohols (methanol and ethanol)
- Organic acids (acetic acid)
- Aldehydes (formaldehyde)
- Embedding: for mechanical support of soft tissues.
• Dehydration – alcohol.
• Clearing – removing of alcohol; xylene.
• Embedding agent: paraffin. - Sectioning: to obtain a thin layer of tissue.
• Microtome.
• Cryostat (for frozen sections) - Staining
- ‘Deparaffinized’ by xylene.
- Rehydrated with alcohol.
- Staining
- H&E
- Covering: reverse process
• Dehydration with alcohol
• Clearing agents – xylene
• Permanent ‘glue’ glass over the section.
What are the compartments of the nucleolus and what are their functions?
1) Fibrillar center (FC) - Pars amorpha.
Contains rRNA genes and NOR (nuclear organizing region)
2) Dense Fibrillar compartment - Pars fibrosa.
Contains actively transcribing rRNA, and snRNP. Pre mRNA modification and processing happens here.
3) Dense granular compartment (DGC) - pars granulosa.
Site of late processing events in the biogenesis of rRNA.
4) Nucleolus associated chromatin.
Heterochromatin
Describe step by step the pathway of lysosomal enzyme from their synthesis to the destination.
Name the cellular components where these steps occur and give the name of the process.
Follows the secretory pathway. Synthesized on the rER like secretory proteins and pass into lumen.
Lysosomal enzymes undergo N-glycosylation in rER then it goes to (cis
Golgi) and being modified by MGP) signal and later on this lysosomal
enzyme will bind to MGP receptor then it forms a vesicle and goes to its
destination. For example: Endosome
The phosphorylation of mannose residues is a criticla step in sorting lysosomal proteins to their correct intracellular destination.
What kind of reaction is the PAS reaction?
What are the main steps of it?
Which structures can be detected by PAS?
- It’s a cytochemical staining method to detect polysaccharides like glycogen. PAS means Periodic Acidic (HIO4) + Schiff reagent (purple). It is the most common step for complex carbohydrate formation (glycogen, glycoprotein, glycolipid).
- Glycogen is everywhere (cytoplasm of liver)
1) Periodic Acid oxidisizes hydroxyl groups into aldehydes of hexoses in complex carbohydrates
2) Schiff reagent (aldehyde detector) gives red colour and red fuchsin precipitation.
What kind of molecules are kinases?
How do they act on their target molecules?
Give at least 3 possible activators of the kinases.
Name 2 different cellular processes that are regulated by kinases.
1) They are enzyme which can phosphorylate its substrate
2)
I. Before the kinase can phosphorylate a signaling protein in the cell, it must first be activated.
II. When signal molecules bind to the two tyrosine kinase receptors, the receptors move closer toghether in the plasma membrane and form a dimer.
III. A conformational change takes place, allowing the kinase part of each receptor to add a phosphate from an ATP molecule to the substrate on the other member of the dimer.
IIII. Once activated, the kinase enzymes can phosphorylate signaling proteins inside the cell.
2) They act on their target molecules by kinase cascade CAMP, Cyclin,
DAG (ciacylglycerol kinase).
4) Mitogen process and conversion between glycogen to glucose
What are the proteosomes and what is their function?
Which proteins and how are they targeted to proteosomes?
Describe how the proteosomes are involved in the regulation of M-phase.
Proteosome is a multienzyme complex. Its function is to degrade the protein.
- Ubiquitin ligases will target proteins to proteosomes so ubiquitin
attaches to the protein lead it to proteosomes - APC is a ubiquitin and it attaches to its substrate and terminate M-
phase (located in the nucleus and cytoplasm)
Detect an enzyme in LM section. Which methods can you choose?
Can you use this method in TEM?
- Histochemistry methods can be used to detect
enzyme - E (enzyme) + P (product) -> E+P (coloured)
- For instance acid phosphates want to be detected. Its substrate is
Naphtyl phosphate. Acid phosphates will cleave off phosphate groups
than niftily is dyed. - Naphtal + salt -> Azodye (quite usable, black)
- No acid phosphate in nucleus in tetrahymena (presence of oxodye
demonstrates enzyme activity) - NO! In TEM we have to use contrast enhancements
What does metachromasia mean?
What is the cause of it?
Give exact examples for metachromasia?
It is a characteristic of certain basic dyes (such as crassly violet,
Toludine blue). When material is stained by them, material gets a colour
different from that dye.
The cationic radicals of the dye bind to the anionic radicals of the substrate in such a way to other the wavelength when the mast cells are stained with toludine blue.
Describe the structure and function of the smooth ER.
In which cells is it found?
It consists of tubules and vesicles (more tubules than rER and it doesn’t
have any ribosomes an the surface)
Function of it changes according to the cell.
- Storage and regulation of Ca2+ for contraction (muscle cells). Sarcoplasmic reticulum.
- Detoxification and breakdown of glycogen(liver)
- Synthesis of steroids, phospholipids, cholesterol, ceramid.
- In every cells: Ca storage
What are the motor proteins? List them.
What are the differences between them?
1) Dyenin + 2) Kinesin: acts on microtubules. Crawls hand-over-hand movement, using its two heads to move forward.
3) Myosin: acts on microfilaments. Crawls using step-by-step inchworm movement.
Both are driven by ATP.
What are the histones?
What are the types of them and what is their function?
Explain what histone code means.
Protein found in eukaryotic cell nuclei package and order DNA into small units nucleosomes. (DNA+histone = nucleosome)
- Histones are classified into 2 groups:
- Nucleosomal Histones (H2A/B, H3, H4)
- HI histones
Function of H2A, H2B, H3 and H4 is forming octomers where DNA is
wrapped on it-
HI histone links the octomers and DNA.
The histone code is a hypothesis that the transcription of genetic information encoded in DNA is partly regulated by chemical modification to histone proteins.
- Histone code is modification of Histone Acetylation, Methylation,
Phosphorylation
Describe the function of SRP (signal recognition particles) in details.
What are the chemical components of it?
SRP is a ribonucleic protein to the endoplasmic reticulum in eukaryotes and the plasma membrane in prokaryotes.
Chemical components of it: RNA and
Proteins.
SRP binds to signal sequence on proteins and it will go to SPP
receptors and protein will go to rER through translocator.
Describe in one sentence the process of apoptosis.
What are the main morphological changes in an apoptosis cell detectable by different microscopic methods?
What is the molecular machinery responsible for apoptosis?
1) Apoptosis is the main process of programmed cell death that may occur
in multicellular organisms.
2) Blebbing, shrinking, apoptotic bodies, chromatin
condensation and fragmentation
3) Proteases - caspases.
What does the cytochemical reaction mean?
What are the basic requirements of this method?
Give at least one example of cytochemical reactions.
This sort of cytological methods combines microscopic and chemical
analytical methods for the localisation of different chemical components
in the cells or tissues
1) reaction product should visible (colour in LM and scattering for EM)
2) For quantitative evaluation precipitate should be proportional to the amount, concentration of the substance being analysed
- The method should be specific for the substrate of the chemical group
that we study
Feulgen Reaction (DNA can be detected)
1) HCL cleaves off the purine bases from DNA then 2 deoxyribose
groups form red colour with Schiff reagent
2) Alderhyde groups form red colour with Schiff reagent
What is the MPF? What are the components of it?
How does it act? Give at least 3 substrates of MPF.
What are the consequences of the changes in the target molecule?
- MPF is M-Phase promoting factor - is a CdK and cyclin complex. It acts
on promoting M-phase.
Consists of nuclear lamins, condensin complex, MAP s(microtubule associated proteins.
Consequences of changes in target molecule:
1) Nuclear lamina will be decomposed.
2) MAP. It can form microtubules, which is needed for the M-phase
3) Activates (indirectly) APC (anaphase promoting complex). It terminates M-phase.
