Cell Signaling I
Describe autocrine cell signaling
A cell will release a ligand that will leave the cell, and the ligand will bind to a receptor protein on the cell surface to activate a signal transduction pathway inside the cell.
What are the three steps in cell signaling and what happens in each step.
1. Reception - ligand (hormone, ion, growth factor) binds to the receptor.
2. Transduction is when binding of the ligand to the receptor protein leads to a series of steps inside the cell where one protein activates or represses another protein in a series.
3. Response - a change in cellular activity ( cell divides or stops dividing, enzyme active or inactive, gene turned on or off, etc.)
1.Describe what happens in the lag, log, stationary, and death phase of bacteria grown in culture.
2. What is the difference between broad spectrum and narrow spectrum antibiotics.
Question 1
Lag phase - bacterial cells getting acclimated to their environment, metabolically active, no increase of the number of cells in the population.
Log Phase- exponential growth of bacterial cells, nutrients are abundant
Stationary Phase - death rate = reproduction rate due to nutrient deprivation, lack of oxygen, changes in pH, waste buildup
Death phase - death rate is greater than reproduction rate due to limiting factors in the environment.
Question 2
Broad spectrum can kill many different species of bacteria, while narrow range can kill only only one particular species of bacteria.
What are the functions of lysosomes
contain hydrolytic enzymes that work at a low pH, break down food, damaged organelles
Daily -Double1. Based on which physical property does agarose gel electrophorese separate DNA
2. 2. What charge does DNA have, why does it have this charge, and in agarose gel electrophorese which electrode will the DNA be attracted to.
1. size
2. Negative charge, due to phosphate group of the nucleotide, attracted to positive (anode).
Describe paracrine, endocrine and gap junctions
Paracrine - signaling between two neighbor cells one cell releases a ligand and then this ligand binds to a receptor on the other cell to elicit a response. Endocrine - signaling between two cells that are far apart one cell releases a ligand and then this ligand travels through the blood stream binds to a receptor on the other cell to elicit a response. gap junctions - connexin transmembrane protein connects to adjacent cells that will allow exchange of materials
What are the two purposes of second messengers.
1. Amplification of the signal 2. Activate a variety of proteins to elicit multiple cellular responses.
1,Describe three structures and their functions that are unique in bacteria
2. What is the difference between bacteriostatic and bactericidal agents
Question 1
1. Pilli and/or fimbriae - short- hair like extensions outside the cell that allow bacteria to adhere to surfaces
2. Nucleoid - region within the cytoplasm where DNA is stored
3. Capsule - located outside the cell wall made of complex carbohydrates that allow bacterial cells to adhere to surfaces and make bacteria more pathogenic by making it difficult to be phagocyted by macrophages
Question 2
Bacteriostatic slows down the growth of the bacteria, while bacteriostatic kills the bacteria.
1. What are four things all cells have in common
2. Four things different between procaryotic and eukaryotic cells
1. cell membrane, ribosomes, cytoplasm, DNA
2. Pro has no nucleus, only eu has organelles, pro smaller, less complex eu larger more complex, pro divides by binary fission, eu by mitosis, pro one small circular chromosome eu many linear chromosomes, pro anaerobic or aerobic, eu aerobic
If you are using thin-layer chromatography to separate amino acids based on their properties of their R groups and the stationary phase is a silica plate that is polar, while you use a non-polar solvent. The three amino acids you are separating are glycine, phenylalanine, and isoleucine. Isoleucine is the most hydrophobic, while glycine is the least hydrophobic. Which amino acid would migrate the furthest, and which one will migrate intermediate, and which one will migrate the least.
Since glycine is the least hydrophobic it will have the strongest attraction to the polar silica plate and migrate the least, while isoleucine since it is the most hydrophobic will have the weakest attraction to the silica plate and will migrate the furthest,
Describe the structure of the ECM and what are two functions of the ECM.
ECM is the connection between transmembrame proteins (such as integrin) with proteins outside the cell such as collagen, fibronectin, and proteoglycans. Functions of the ECM are 1. Anchor the cell 2. Cell signaling
Explain in detail how intracellular receptors work including describing the size and charge of the ligands
A small/ non-polar ligand will cross the phospholipid bilayer will enter into the cell and bind to the receptor inside the cell which will then elicit a transduction cascade that will lead to a cellular response.
Daily Double -Compare and contrast gram -positive and gram- negative bacteria
2. Explain how you perform gram staining and how does this allow you to distinguish gram-positive and gram- negative bacteria
Both made of peptidoglycan. In gram pos. there is a thick peptidoglycan layer outside the cell membrane. In gram neg. the peptidoglycan layer is thinner and is located between the outer and inner cell membrane, which is unique to gram neg. bacteria.
2. Cells are treated with crystal violet, which will cross both the cell wall and cell membrane and both gram positive and gram neg. will appear violet. Cells are then treated with iodine, which will cross both the cell wall and cell membrane and both gram positive and gram neg. and bind to the crystal violet. Cells are then treated with alcohol which will be able to only penetrate the thinner gram neg. cell wall and then enter across the cell membrane and wash away the crystal violet stain inside the cell. At this point gram positive will still appear violet, while gram neg will now be colorless. Safranin is then applied and gram positive will still appear violet, while gram negative will appear pink.
What must happen to a polypeptide in the lumen of the rough ER and if these processes occur where does the protein go next.
Both N-linked glycosylation and chaperones help polypeptide chain to fold into a protein and if folded correctly will travel to the golgi.
Explain the purpose of the Calcium chloride and the heat shock in a bacterial transformation.
2. If you transform a bacteria strain that is both ampicillin and kanamycin sensitive with a plasmid that contains a kanamycin resistance gene explain if it will grow or die on the following plates
A. LB w/o antibiotics
B. LB + AMP
C. LB + Kan
D. LB + AMP + Kan
1. Positive charge Calcium ion shields the negative charged DNA, heat shock opens up the cell membrane so the plasmid can enter the cell.
2. A. survive because plate contains no antibiotics
B. Die because plasmid only contains Kan resistance gene, will there fore still be sensitive to AMP
C. Survive because plasmid contains a Kan resistance gene
D. Die because plasmid only contains Kan resistance gene, will there fore still be sensitive to AMP
Describe in detail how an ion- channel linked receptor is activated.
An ion acts as the ligand binds to the ion channel receptor and this will cause a conformational change in the ion channel linked receptor that will either open or close the chanel, therefore either allowing or preventing ions to enter or exit the cell.
What are two examples of second messengers.
Ca+, cyclic AMP, DAG
1. Describe four features a plasmid must have for cloning purposes
2. Describe how conjugation occurs between two bacteria and provide an example how conjugation provides a selection advantage for bacteria
Question 1
1. Origin of replication - so there are numerous copies of the plasmid int he host cell, therefore there will be ample production of the protein of interest
2. Selection Marker - ex. antibiotic resistance gene so it can be differentiated, which cells were successfully transformed into the host cell.
3. Multiple cloning site (MCS) - a region on the plasmid that contains a vast selection of restriction sites where the gene of interest can be cloned into
4. Promoter - upstream of the gene of interest so RNA polymerase knows where to start transcription
Question 2
The bacteria cell that contains the plasmid is F+, while the bacteria cell that does not contain the plasmid is F-. A pillus connects the F+ and F- bacteria, the plasmid in the F+ bacteria replicates and a copy of the plasmid is transferred from the F+ to the F- converting the F- into a F+. Since a new gene(s) are introduced into the F- this could provide a selection advantage for the F- as it can acquire for an example an antibiotic resistance gene or a gene to make an essential nutrient.
Describe in detail the modifications and how the Golgi determines the final destination of a protein
1.Protein travels from rough to cis golgi, where modification of N-linked glycosylation occurs
2. O-linked glycosylation occurs in medial golgi
3. Trans golgi determines the destination of the protein, if a mannose-6 phosphate is added to the protein it will go to the lysosome, if not then woll be secreted out of the cell
. Suppose with affinity chromatography after the wash buffer step you find the protein of interest is eluted. Provide two possible scenarios this could of happened.
2. What would be the ideal item to use in the stationary phase in affinity chromatography if you want to isolate one specific protein
1. Wash buffer is too stringent eluted the protein of interest off, protein of interest had a non-specific interaction with the bead/matrix on the stationary phase.
2. An antibody that recognizes the protein of interest.
1. Ligand binds to the the receptor and the RTK dimerize.
2. Once the RTK's dimerize autophosphoryl ation of the intracellular domains of the the tyrosine residue on each dimer subunit.
3. The phosphorylated tyrosine's will attract other proteins inside the cell and bind them and activate them by adding a phosphate group to these proteins. Since the phosphorylated RTK attracts many different proteins many different cellular responses are elicited.
Explain in detail how a G-protein linked receptor is activated.
1. A ligand binds to G-protein linked receptor. 2. When ligand binds to G-protein linked receptor this results in a conformation change that will result in the G-protein receptor will bind to the G protein and cause the G protein to exchange GDP for GTP. 3. When the G protein is bound to GTP it is active and activate enzymes that will produce second messengers such as cyclic AMP, IP3 which will cause Ca+2 from smooth ER which CA+2 act as a second messenger, DAG that will allow an amplification of the signal and activation of a lot of different proteins to elicit an array of responses
1. Describe five ways antibiotics kill bacteria
2. Describe five ways bacteria become resistant to antibiotics
Question 1
1.Inhibition of cell wall synthesis
2.Breakdown of the cell membrane structure or function
3.Interference with functions of DNA and RNA
4.Inhibition of protein synthesis
5.Blockage of key metabolic pathways
Question 2
1. Drug inactivation
2. Decreased permeability
3. Drug is pumped out of the cell
4. Change in binding site
5. Use an alternative pathway
1. mRNA initially translated on a ribosome in the cytoplasm.
2.A signal sequence, which is 20 hydrophobic amino acids is recognized by the SRP.
3. SRP directs the ribosome/polypetide/mRNA complex to a SRP receptor on the rough ER.
4. The ribosome/polypetide/mRNA is transported to a translocon protein, where translation continues, and the polypeptide chain is threaded into the lumen of the ER
. If you are doing ion exchange chromatography and you want to perform cation exchange the proteins of interest have a Pi 7 and 9, What pH must the equilibration buffer be.
2. If you wanted to separate three different proteins: Protein A has a pI of 5, Protein B has a pI of 7 and Protein C has a pI of 8.5. If these proteins were mixed in a buffer that is 8 which of these protein(s) could you use anion exchange chromatography to separate.
1. The pH of the buffer must be less that pH 7 so the proteins will have a positive charge.
2. Protein A and protein B because they would have a negative charge