Objectives A Comprehensive Comparison Of Transmembrane Domains Reveals Organelle‐Specific Properties Hydropathy plots • Explain hydropathy plots • To obtain a clear picture of organelle‐specific restraints on TMDs • Perform comprehensive comparisons of the TMDs of proteins in different organelles Method Transmembrane domain BLAST, NCBI. http://blast.ncbi.nlm.nih.gov/Blast.cgi Why were hydropathy plots used? 1) To show if proteins were hydrophobic or hydrophilic 2) To show the length of average protein TMDs in different membranes 3) To indicate the width of the membrane 4) To identify proteins Hydrophobicity Values: free energy required for transfer from water to a hydrophobic environment • Negative value(‐): the TMD has a preference for the interior of the lipid bilayer • Positive value(+): hydrophilic residues hydropathy index Approximately how many amino acid residues long is a PM TMD? a) 17 residues b) 19 residues c) 20 residues d) 24 residues e) 26 residues Membranes can have different widths What can you conclude from the data? a) Proteins with TMDs having a mean residue length of 24 are found in the plasma membrane b) The average length of a group of TMDs corresponds with the width of the membrane they are located in c) As you move further from the cystolic edge, TMDs become more hydrophobic d) Plasma membranes are wider than ER membranes Organelle membranes do not necessarily have the same lipid composition Molecular Biology of the Cell, 5th ed. Table 10‐1 Transmembrane domains (TMDs): • Sequences of TMDs of integral membrane proteins should reflect the physical properties of the bilayers in which they reside. • TMDs are not generic but have organelle‐ specific properties • TMDs of proteins in the ER and Golgi are shorter than the TMDs of plasma membrane proteins, thus the ER and Golgi have thinner width membranes References • A Comprehensive Comparison of Transmembrane Domains Reveals Organelle‐Specific Properties Hayley J. Sharpe, Tim J. Stevens, and Sean Munro Cell. 2010 July 09; 142(1): 158–169 http://www.ncbi.nlm.nih.gov/pmc/articles/P MC2928124/ • BLAST, NCBI. http://blast.ncbi.nlm.nih.gov/Blast.cgi • Molecular Biology of the Cell, 5th ed. Figs 10‐21, 10‐ 12, Table 10‐1 If two amino acid signal sequences on a single protein were expressed, where would the protein end up? All eukaryotic cells have peroxisomes. Cells were engineered to not contain peroxisomes. • How can peroxisomes be visualized in a cell?  Signals for import into the nucleus and import into the ER:  Signals for import into mitochondria and retention in the ER: • Why does the fluorescence show up as small dots in normal cells? normal cells • Would peroxisomal proteins still be produced in cells that do not contain peroxisomes?  Signals for import into the nucleus and export from the nucleus: peroxisomedeficient cells Which kind of translocation inserts proteins into the ER? • If so, where are peroxisome enzymes located in peroxisome‐deficient cells? For the following soluble proteins, what would happen if… 1. You add a signal sequence for the ER to the amino‐ terminal end of a normally cytosolic protein: 2. You change the hydrophobic amino acids in an ER signal sequence to charged amino acids: 3. You change the hydrophobic amino acids in an ER signal sequence to other hydrophobic amino acids: 4. You move the amino terminal ER signal sequence to the carboxy‐terminal end of the protein: Number of membrane spanning segments? Post‐translational translocation is used: a) b) c) d) e) f) g) For proteins targeted to the nucleus For proteins targeted to the mitochondria For proteins targeted to peroxisomes For proteins targeted to the ER When ribosomes are attached to the ER For proteins targeted to the Golgi For proteins targeted to lysosomes Timeline Stem cells 1962 SCNT (somatic cell nuclear transfer) 1996 Dolly 1981 mouse ESC (embryonic stem cells) 1998 human ESC 2001 President G.W.Bush 2006 mouse IPC (induced pleuripotent stem cells) 2007 human IPC 2009 President Obama 2017 President Trump 2021 President Biden Hair follicles (red) grow radially out of spherical skin organoids (from mouse IPCs) JIYOON LEE AND KARL KOEHLER What is the source of ESC? stemcells.nih.gov Nuclear Import of Proteins Objective: to determine the function of each of the components of the Ran GTPase system Nuclear import and export are often studied in cells treated with mild detergents under carefully controlled conditions, so that the plasma membrane is freely permeable to proteins, but the nuclear envelope remains intact. Because the cytoplasm leaks out, nuclei take up proteins only when the cell remnants are incubated with media that is a source of critical soluble proteins. Nuclear transport can be followed by labeling the protein of interest (a substrate that contains a nuclear localization signal) with a fluorescent tag. The small GTPase, Ran, was shown to be required for nuclear uptake by using this assay. It requires GTP for activity. Neither Ran-GDP nor Ran-GTP bind to nuclear localization signals so other factors must be responsible for identifying proteins to be imported into the nucleus. Fig. A shows positive and negative controls for the experiment in Fig. B. Solid circles represent nuclei containing the fluorescently labeled substrate. Outlined circles represent accumulation of substrate at the nuclear periphery. Importin is a newly purified protein for which you need to assign a function. • • The crude cytosol contains everything required for nuclear uptake of the labeled substrate. Is GTP essential for nuclear uptake? • Is Ran + GTP able to promote nuclear uptake of the labeled substrate? • Does importin promote nuclear uptake? Explain. • What are the essential components for nuclear uptake of substrates? Why do you think the substrate accumulates at the nuclear periphery, as it seen in the absence of GTP or with importin alone in the presence of GTP? The data in Fig. B were generated to determine the steps in the uptake pathway. Nuclei were first incubated with substrate in the presence of importin. Unbound importin and substrate were washed away and then the nuclei were incubated with Ran and GTP. • From these data, decide whether importin is a: ➢ nuclear import receptor ➢ Ran-GTP ➢ Ran-GEF Explain your reasoning. • Using Fig. 15-10, what steps in the import process can be explained by this data? How does a protein know if it should be translated on the RER or a free ribosome? Which proteins are synthesized on free ribosomes? a) b) c) d) e) f) Cytosolic proteins Nuclear proteins Mitochondrial proteins Secreted proteins Plasma membrane proteins Lysosomal proteins = Table 15‐3 ECB4e A N‐terminal signal sequence would be used for: a) b) c) d) e) Proteins targeted to the nucleus Proteins targeted to the ER Proteins targeted to the mitochondria Proteins targeted to the golgi Proteins targeted for secretion Can a protein translated on a free ribosome be secreted? Fig. 12-6 MBOC 5e What is the fate of a protein with no sorting signal? a) b) c) d) e) Nucleus Cytosol Mitochondria Secreted outside the cell Plasma membrane What is true about gated transport into the nucleus? a) The nuclear membrane is freely permeable to ions and other small molecules IP3 > PKA > effect? E2 > PKA > IP3 > effect? Conclusions • E2 increases PKA activity which increases IP3 levels • Adenylyl cyclase pathway comes before phospholipase C pathway in the nongenomic signaling of oviductal transport References • Alberts et al. Fourth Edition Essential Cell Biology. 2014 Print. • Sci Vis Lab. “Estrogen Receptor (II): Molecular and Cellular Receptors.” Online video clip. YouTube. YouTube, 15 Jan. 2008. Web. 5 Dec. 2015. • Losel R & Wehling M 2003 Nongenomic actions of steroid hormones. Nature Reviews Molecular Cell Biology 4 46‐56. • Orihuela, P et al 2006 Inositol triphosphate participates in an estradiol nongenomic signalling pathway involved in accelerated oviductal transport in cycling rats. Journal of Endocrinology 188, 579‐588 What would be considered to be an amplifier protein? What would be considered a transducer protein? a) Adenylyl cyclase b) Cell surface receptor c) cAMP a) b) c) d) What would be considered to be a small intracellular mediator? a) b) c) d) e) Integrating proteins would: a) Combine different signals and send out a single signal b) Combine different signals and send out several signals PKA cAMP PLC IP3 Ca2+ Which type of signaling mainly works via ion channel‐coupled receptors? What is NOT characteristic of ion channels? a) A type of carrier b) Hydrophilic pores spanning the membrane c) Transport more molecules faster than carriers d) Can be active or passive e) Found in all membranes f) Highly selective Adenylyl cyclase cAMP IP3 Ion channels a) b) c) d) Growth factors Peptide hormones Steroid hormones Neurotransmitters Which type of signaling works via enzyme‐ coupled receptors? a) b) c) d) Growth factors Peptide hormones Steroid hormones Neurotransmitters Which kinase phosphorylates on a tyrosine amino acid residue? a) b) c) d) e) PK‐A PK‐C PDGF receptor MAPK CaM kinase What type of effects do growth factors have? a) b) c) d) e) Protein synthesis Proliferation Angiogenesis Motility Cell survival What is NOT characteristic of ion channels? a) A type of carrier b) Hydrophilic pores spanning the membrane c) Transport more molecules faster than carriers d) Can be active or passive e) Found in all membranes f) Highly selective Which signaling pathway would react most quickly (seconds) to environmental changes? a) b) c) d) Classic steroid hormone pathway G‐protein linked pathways Tyrosine‐kinase pathways Ion channels Most steroid hormone effects are considered to be: Cell Communication a) Genomic b) Non‐genomic Clicker slides Characteristics of molecules requiring cell surface receptors: a) Hydrophobic b) Requires a secondary messenger inside the cell c) Uses preexisting machinery to exert its effect d) Causes gene transcription What are the two main signaling proteins that act as molecular switches? a) b) c) d) Calcium GTP binding proteins Kinases Transcription factors Which type of signaling works mainly via G‐ protein‐coupled receptors? a) b) c) d) Growth factors Peptide hormones Steroid hormones Neurotransmitters A characteristic of a signaling cascade is: a) Multiple amplification steps b) Several relay proteins that pass the signal on c) One protein activates the next in the cascade A fast (and effective) signaling system requires: a) Lots of stimulus (i.e. chemical messenger) b) Amplification c) Low resting concentrations of intermediates (mediators) d) Rapid on/off intermediates e) Readily activated degradation systems (e.g. PDE, pumps, phosphatases) …machinery that the receptors are hooked up to Do the βγ subunits do anything? How does a cell know how to respond to extracellular chemical messengers? • chemical messengers present • receptors expressed • machinery the receptor is hooked up to • signaling pathways integrated Quick Physiology Muscle Induces Neuronal Expression of Acetylcholinesterase in Neuron‐Muscle Co‐culture Dhwani Patel, Brandon Nguyen, Dr. Wilson Neurons communicate by releasing neurotransmitters at neuromuscular junctions (synapse in picture below).  After a neurotransmitter is released it needs to be removed. • Can be accomplished by a degrading enzyme i.e. Acetylcholinesterase (AChE) Overall Objective of Paper To determine whether muscle-induced AChE expression in motor neurons is mediated by intracellular cAMP signaling pathway Experimental Objective To determine whether CREB phosphorylation increases in response to addition of muscle extract which would indicate if cells are mediated by cAMP signaling pathway How CREB works • CREB = Transcription Factor/ Transcription regulator o Activated CREB binds to cAMP Response Element (CRE). 1. Signal molecule CGRP binds to G-protein Coupled Receptor → Activates GPCR 2. Active GPCR → activates 𝛼-subunit of G-protein 3. Active 𝛼-subunit → Activates Adenylyl Cyclase 4. Active Adenylyl Cyclase → converts ATP to cAMP 5. cAMP → activates Protein Kinase (PKA in diagram) 6. Protein Kinase → phosphorylates CREB! 7. Active CREB → binds to CRE to initiate AChE gene transcription in vitro simulation of neuromuscular junction o Coculture: • NG108-15 cells (motor neuron) were stabily transfected with AChE gene promoter • chick myotube (muscle cell) o In this experiment- NG108-15 neural cells were treated with a muscle extract or pharmacological agents o CREB and pCREB detected by Western blotting o cAMP detected by ELISA Clicker Question: Which of these will induce cAMP? A. CREB B. GDP C. Forskolin D. Bt2-cAMP E. AChE Factors Involved 1. cAMP → intracellular signaling molecule generated from ATP by adenylyl cyclase Results Figure 7A 2. Cyclic AMP Response Element (CRE) → Region in the promoter of AChE gene that active CREB binds to promote AChE gene transcription ● Cultured NG108-15 cells treated with muscle extract 3. Cyclic AMP Response Element Binding Protein (CREB) → Transcription factor that binds CRE region on promoter when activated by Protein Kinase A ○ The amount of cAMP induced increases and then decreases 4. Forskolin: lipid soluble compound that activates adenylyl cyclase ∴ increases intracellular levels of cAMP  Peak activation at 5-30 minutes. 5. Dibutyryl-cAMP (Bt2-cAMP) → lipid soluble compound; mimics effect of cAMP by activating Protein Kinase and is resistant to phosphodiesterase (PDE) degradation Clicker Question: Results Based on the observation that in the muscle-extract treated neurons, phosphorylation of CREB peaks after 10 minutes whereas cAMP generation peaks after 5 minutes, what is a valid conclusion from Figures A and C? Cultured Neurons were treated with the following: 1. Muscle Extract A. cAMP generation is dependent upon phosphorylation of CREB. o P-CREB levels upregulated at 5 min and returned to normal after 1hr B. Phosphorylation of CREB is dependent upon the intracellular levels of cAMP. 2. Bt2-cAMP C. Phosphorylated CREB was expressed first in cell cultures exposed to o P-CREB was not upregulated until 30 min after activation and stayed elevated 3. Forskolin o P-CREB upregulated at 5 min and stayed elevated Bt2-cAMP. D. Activation of the CREB phosphorylating kinase is dependent upon Immunoblot assay Antibodies used to identify phosphorylated CREB and total CREB intracellular levels of cAMP. References Conclusion 1. 2. Muscle-induced AChE expression in NG108-15 cells via an intracellular cAMP signaling pathway is evident by increased phosphorylation of CREB. Spike of cAMP prior to increase in P-CREB indicates that the phosporylation of CREB is dependent upon cAMP. • Alberts, B. (2014). Essential cell biology (4th ed., pp. 527, 546-547). New York: Garland Pub. • Forskolin from Coleus forskohlii, ≥98% (HPLC), powder | Sigma-Aldrich. (2016). Sigmaaldrich.com. Retrieved 11 April 2016, from http://www.sigmaaldrich.com/catalog/product/sigma/f6886?lang=en®ion=US • Sp-Adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt hydrate ≥98% (HPLC), solid | Sigma-Aldrich. (2016). Sigmaaldrich.com. Retrieved 11 April 2016, from http://www.sigmaaldrich.com/catalog/product/sigma/a166?lang=en®ion=US • Jiang, J., Choi, R., Siow, N., Lee, H., Wan, D., & Tsim, K. (2003). Muscle Induces Neuronal Expression of Acetylcholinesterase in Neuron-Muscle Co-culture: TRANSCRIPTIONAL REGULATION MEDIATED BY cAMP-DEPENDENT SIGNALING. Journal Of Biological Chemistry, 278(46), 45435-45444. http://dx.doi.org/10.1074/jbc.m306320200 • https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biologylearning-center/protein-biology-resource-library/pierce-protein-methods/overviewelisa.html Objectives I Cell Disease Dataset • Determine whether total lysosomal enzyme supplementation method can work on lysosomal storage disorders. • Determine the effect of M6P on lysosomal enzymes Mohamad Odeh, David Beecher, Naeem Ahmed Background Info ML‐II: Metabolic disease caused by deficiency of GlcNAc‐ phosphotransferase. Lysosomes of the patients lack dozens of enzymes targeted by the M6P receptor‐dependent pathway and they show developmental delay and severe bone deformities. • GlcNAc‐Phosphotransferase: Plays a role in generating M6P recognition markers on lysosomal enzymes • M6P: Receptor that plays a role in transporting proteins from the Golgi and cell surface to lysosomes • LAMP‐2: Lysosomal associated membrane protein • Cathepsin B: Lysosomal enzyme CQ 1 Background Info (cont'd) • Lysosomes: Involved in degrading and recycling cellular waste, cellular signaling, and energy metabolism • ML‐II impairs the Mannose‐6‐phosphorylation (M6P) of lysosomal enzymes • This will cause an accumulation of undigested substrates in lysosomes Methods • Cell lines and cell culture 1. Obtained skin fibroblasts from 3 unaffected and 3 individuals affected with ML‐II • What is the effect of NH4Cl on normal cells? a.) Increased secretion of lysosomal enzymes b.) Decreased secretion of lysosomes c.) No effect d.) Increased secretion of lysosomes e.) Decreased secretion of lysosomal enzymes • Florescence staining and micrography cells 1. Images were acquired using fluoresence microscope or confocal laser scan microscopy system 2. Looked at all skin fibroblast cell lines and confirmed similarities within each group • Enzyme Treatment 1. Treated normal cells with NH4Cl to cause secretion of normally tagged lysosomal enzymes 2. These were collected and used to treat MLII cells to rescue the phenotype • Western Blot 1. Used to visualize lysosomes and the enzymes in the lysosome • Measurement of lysosome amount in each cell 1. The LysoTracker/DAPI intensity ratio was calculated – this estimates the lysosomal amount (number and size) in each cell Data Interpretation 1 • ML‐II Cells secreted more enzymes than normal cells • Normal + NH4Cl cells had similar secretion profiles to ML‐II cells • M6P tag is not present in ML‐II cells which is why they secreted more enzymes via constitutive exocytosis M6P Receptor Pathway Data Interpretation 2 • Shows localization of enzymes in the cytoplasm within normal, ML‐II, and enzyme treated ML‐II skin fibroblasts • Merging LAMP‐2 and cathepsin B shows that incorporated enzymes were targeted to lysosomes • ML‐II cells that had enzymes added were able to uptake those enzymes and direct to the lysosome. Data Interpretation 3 CQ 2 • Did total enzyme supplementation treatment on ML‐II cells work? a.) Yes, lysosomal enzymes were able to enter lysosomes and degrade cellular debris b.) No, lysosomal enzymes were not able to enter lysosomes c.) Yes, lysosomal enzymes were exocytosed back into lysosomes d.) No, the cells underwent apoptosis Conclusion • LysoTracker is used to show presence and function of lysosomes. DAPI fluorescence stained the nuclei. • Lysosomal amount is higher in ML‐II cells than normal cells. The lysosomes aren't functioning properly since there are no lysosomal enzymes in order for them to. Remember lysosomes are used for degradation of cellular debris. • Lysosomal amount was decreased after enzyme treatment. This means that lysosomal enzymes are entering the lysosome and lysosome is functioning again. • M6P receptor is integral to lysosomal enzymes transport into lysosome. • Total lysosomal enzyme supplementation method ( enzyme treatment) helped return lysosomal enzyme activity back to normal • Excessive lysosomal storage materials are the cause of ML‐II Objective: To order the components in a cell signaling pathway based on protein mutation data. In a hypothetical pathway, Ras, protein X, and protein Y are all required for proper signaling. • Cells in situation (A) were mutated so they do not express protein X. They still express protein Y. • Cells in situation (B) were mutated so they do not express protein Y. They still express protein X. • “Overactive Ras” means that Ras was transfected into the cells and is overexpressed. 1) Make an observation and corresponding conclusion for situation (A) and situation (B). Alternatively, state the conclusion based on the evidence. The transfected cells with Ras and mutant to protein X showed an activation and a restored signal which means that X is necessary for the signal transmission to the Ras. CONCLUSION: PROTEIN X ACTS UPSTREAM OF Ras The transfected cells with Ras and mutant to protein Y showed a negative result and no activation upon Ras activation which means that Y is necessary for the signal transmission after Ras. CONCLUSION: PROTEIN Y ACTS DOWNSTREAM OF Ras 2) Use your conclusions (above) to order the pathway starting with the receptor (RTK). Your pathway must include Ras, protein X and protein Y. (A) When protein Y is inactivated, no signaling occurs in the cells. Furthermore, when introducing a hyperactive form of Ras, the signaling can be bypassed, allowing the pathway to function even when the extracellular signal molecule is absent. Protein X appears to act upstream of Ras. (B) When protein Y is inactivated in cells, signaling is disturbed. The pathway order is: RTK → X → Ras → Y 3) Why was a signal molecule not necessary to cause a response (“signaling restored”) when Ras was transfected into the cells in situation (A)? The signaling molecule produce in the downstream pathway the activation of Ras, and the downstream response due to Ras activation. When cells are transfected with overactive Ras, this mimics the signal molecules and therefor no extracellular molecule is needed to reproduce the pathway activation. ➢ In the cAMP/ p-CREB dataset presented in class, what was the condition that did not require a signal molecule to cause a response? (see backside) Explain how the treatment worked without a signal. The condition that did not require a signal molecule is the condition with Bt2-cAMP. I fact the cAMP activation is a downstream effect of the extracellular signal and therefor the activation of cAMP mimics the signal molecule. The Forskolin is a lipid soluble compound that mimics cAMP by activating the downstream molecule which is the Protein Kinase and therefor produce phosphorylated CREB and the downstream effect. Packet includes: ▪ Field leaps forward with new stem cell advances. Science Vol 318, 23 Nov 2007 ▪ The quest for the perfect reprogrammed cell. Nature Vol 511, 3 July 2014 ▪ Stem Cells Made Waves in Biology and Medicine, The Scientist 1 October 2016 ▪ Pluripotent stem cells progressing to the clinic. Nature Reviews Vol 17, March 2016 ▪ Child Receives Transgenic Skin over Most of his Body. The Scientist 8 Nov 2017 ▪ Vision Loss after Intravitreal Injection of Autologous “Stem Cells” for AMD Other resources: ▪ Info about current initiatives in the stem cell field: https://stemcells.nih.gov/ ▪ ECB4e Ch 20: pp.708-712 ▪ On D2L: Cardiologist trades stem cells for cell-based meats (Memphis Meats) Lancet, 2018 and other interesting links ▪ “Stem Cell Basics. In NIH Stem Cell Information. https://stemcells.nih.gov/info/basics ▪ https://www.stemcell.com/technical-resources/area-of-interest/organoid-research.html 1. Use the Stem Cell Basics website to describe the differences between totipotent, pleuripotent and multipotent stem cells. ➢ Pluripotent stem cells are stem cells that that are capable of becoming into all cells of the body. Multipotent stem cells are more specialized and the type of cell they develop into is more limited. An example would be an adult stem cell as it is limited to the type of cell it can be (NIH, 2016). The totipotent cell would then be the embryo, the cell that can give rise to an organism and by extension the trophectodermal cells that support the placenta. 2. Describe the origin and characteristics of embryonic stem cells (ES cells). ➢ Embryonic stem cells are the cells found in the inner cell mass of a human embryo in the blastocyst stage. These cells are of great interest to scientists, because unlike nerve cells, muscle cells and blood cells, they can self-renew and replicate multiple times (NIH, 2016). They are also undifferentiated, meaning that they have the capacity to be any cell. Tissuespecific characteristics are absent in these cells, which means that they cannot perform any specialized function. 3. Describe the origin of and characteristics of adult stem cells. ➢ Adult stem cells are the repair system of the various parts of the body. They become activated if the body feels that there is a need to replace damaged or old cells. They are generally associated to specific organs and parts of the body (NIH, 2016). 4. Describe the origin of and characteristics of induced pleuripotent cells (iPCs). ➢ Scientists in 2006 discovered that under certain conditions, they could reprogram mature human adult cells into a more embryonic stem-like form by forcing expression of genes that are regulators of pluripotency. These are then called induced pluripotent cells (NIH, 2016). 5. c-myc is considered to be an oncogene. What is a possible drawback for using this particular gene to create iPCs? ➢ An oncogene is a gene that can possibly transform a cell into a tumor, which is perilous if the plan is to integrate it into therapy or other uses as it can become cancer. 6. Describe how iPCs are different from ES cells. ➢ iPCs are formerly adult human cells that were reverted into a more pluripotent-state, which means that they were once embryonic cells that already differentiated and was then reprogrammed. ES cells are “fresher” meaning that they came straight from the inner cell mass of a human embryo and therefore, have not yet differentiated (NIH, 2016). 7. What is the main advantage of using iPCs over IVF (in vitro fertilization) ES cells? ➢ Knowing where both these cells come from gives an insight on the main advantage of iPCs, which is its accessibility. iPCs can just be taken from mature human cells and genetically match its source. On the other hand, IVF ES cells would require creating human embryos, which has very ethical issues and any research involving it would be under heavy scrutiny and it would not be cost-effective as numerous egg cells would be needed. 8. What are the advantages and disadvantages of generating ES cells using somatic cell nuclear transfer (SCNT)? ➢ Since it only requires a somatic cell and an egg cell, SCNT bypasses some of the ethical issues deriving from the traditional acquisition of ES cells. The chances of cancer are also lower since it does not involve the introduction of certain transcription factors. Also, according to Krupalnik & Hanna (2014), the reprogramming process in SCNT is much more natural as it is more comparable to the process IVF ES cells undergo. ➢ There are still a few problems such as DNA-Methylation defects. There is also the ethical concern of human cloning and the fact that the chances of failure are high for this process means that a large number of egg cells must be donated (Krupalnik & Hanna, 2014). 9. Describe how iPCs are different from adult stem cells. ➢ iPCs are former adult cells, but they were reprogrammed in such a way that they reverted into a more embryonic form and can be differentiated into other cells. Adult stem cells are much more specialized or multipotent and can only transform according to their specialization (the blood stem cells found in the blood marrow can only become blood cells) (NIH, 2016). 10. Are iPCs currently used for treating diseases? ➢ The technology is still new, but in 2014, Takahashi in collaboration with Yamanaka began an iPC clinical trial for a therapy of macular degeneration. It was put on hold due to the occurrence of mutations in the cell but one patient successfully received the treatment (Zusi, 2016) 11. Do a quick search, are ES cells or adult stem cells currently being used for treating diseases? ➢ There are multiple ways adult stem cells are being used to treat diseases. The most well-known would be blood marrow transplants. Hemopoietic progenitor cells from cord blood is also being used to treat blood diseases and is FDA approved (NIH, 2016). A child who was suffering from epidermolysis bullosa underwent gene therapy. Keratinocytes from the undamaged skin were taken and grown in culture and was then transduced using a retroviral vector that carried a healthy version of the laminin b3 coding sequence (the cause of the disease was a mutation in this gene). The skin grew in sheets and was transplanted to the boy. Two years later, the boy’s skin is still healthy (Williams, 2017) 12. Which type of stem cell is best to use for treating disease? What needs to be addressed before any of these stem cells are widely used? ➢ The most promising would be iPCs as they do not need human embryos, only adult stem cells of which there are plenty. Unfortunately, when these are compared to normal embryonic stem cells that arise during normal embryonic development it is apparent that refinements to the reprogramming process are needed (there are still epigenetic differences between iPCs and normal ES cells). This needs to be addressed as they may cause mutations that severely affect the patient (Krupalnik & Hanna, 2014). 13. The technology to grow ES cells in culture was developed in 1998. What was the main limiting factor in ES cell research up until 2009? What is the situation today? ➢ The main problem was the backlash that came after the revelation that human embryos were being utilized in stem cell experiments. In 2001, President Bush, decided to prohibit federal funding for human stem cell lines. Fortunately, in 2009, the NIH approved federal funding for 13 human stem cell lines. Today, multiple clinical trials have been held or are being held. Examples are spinal cord therapies, treatments for macular degeneration, generation of pancreatic beta cells for diabetes and more (Zusi, 2016). 14. p53 was found to limit iPC generation. Why would suppressing p53 enhance the efficiency of iPC generation? Why would it be appropriate for p53 be added back to the iPCs after they have been made? ➢ That is because the process of reprogramming focuses on gene expression of oncogenes (Vogel & Holden, 2007); and since p53 regulates the processes involving DNA damage and cancer, suppressing it will allow reprogramming. But since it regulates DNA damage, it would be needed once more to stop mutations in the cell (Alberts et al., 2019). 15. Define an organoid. What is it made from? What kinds of things might organoids be used for? ➢ An organoid is a three-dimensional cell culture and has similar features of its represented organ. They can provide physiological model since they are a bit more similar to an organ in vivo, which is also why they are important as they can be used to study the effects of a disease or how an organ reacts to a certain drug (NIH, 2016; “Organoid Research,” n.d.) References Alberts, B. (2019). Essential cell biology (5th ed.). W.W. Norton. National Institutes of Health. (2016). Stem cell basics. https://stemcells.nih.gov/info/basics/stcbasics/#stc-IV Krupalnik, V., & Hanna, J. H. (2014). The quest for the perfect reprogrammed cell. Nature, 511(7508), 160–162. https://doi.org/10.1038/nature13515 Organoid research. (n.d.). Stemcell Technologies. Retrieved December 3, 2021, from https://www.stemcell.com/technical-resources/area-of-interest/organoid-research.html Vogel, G., & Holden, C. (2007). Field leaps forward with new stem cell advances. Science, 318(5854), 1224–1225. https://doi.org/10.1126/science.318.5854.1224 Williams, R. (2018, April 17). Child receives transgenic skin over most of his body. The Scientist Magazine®. https://www.the-scientist.com/daily-news/child-receives-transgenic-skinover-most-of-his-body-30642 Zusi, K. (2018, June 12). Stem cells made waves in biology and medicine. The Scientist Magazine®. https://www.the-scientist.com/features/stem-cells-made-waves-in-biologyand-medicine-32778 Analyzing the Hydropathic Plots Dima Hamad, Mohamad Hamad, and Abdullah Ahmad Objectives ● ● ● Understand the hydrophobic and hydrophilic tendencies of an amino acid sequence Determine what matters in a hydrophobicity plot Explain how the hydrophobicity plot is used Transmembrane Domain - It consist of nonpolar amino acid residues, transverse bilayer contain α-helical transmembrane domains (TMDs), span the hydrophobic core The primary constraint on all TMDs that enter the secretory pathway Sec61 is a membrane protein complex Secretory pathway Translocon - Residues were modeled on an α helix. Hydrophobic residues are colored cyan, polar residues) orange, and basic residues are colored in red. Thicker transmembrane domain: longer TMD and saturation Blast - Blast is used for comparing sequencing information Reference proteins Orthologous proteins What is important in hydrophobicity plot? -type of amino acid - Position/organelle its taking place - length of protein membrane and saturation https://www.youtube.com/watch?v=frVoetPCMWY Clicker Questions What happens if the lipid transmembrane domain is thicker/wider? A. B. C. D. The proteins will have longer TMDs, The line on graph will take longer to decrease The proteins will have longer TMDs, the line on graph will decline quicker The protein will have shorter TMDs, the line on graph will take longer to decrease There is no difference - This graph is a hydrophobicity graph This hydropathy plot allows for visualization Y-axis, x-axis Line at zero Above and below 0 Data Interpretation - Starting at 0 Travels to hydrophobic phospholipid bilayer lumen Golgi vs. Plasma membrane -Golgi decreases first - golgi has 20 amino acids - plasma membrane has 24 What is a possible conclusion for this dataset? A. B. C. The plot display the distribution of polar and nonpolar residues along a protein sequences, therefore the golgi TMD has the thickness and widest membrane causing it to decrease at a very slower rate than the rest plot can be used to predict the transmembrane helices from the amino acids indicated, the type of amino acid, position of where it takes place and thickness of lipid membrane domain contribute to how the hydropathy plot results will turn out plot can identify the membrane spanning segments of a beta barrel, which causes the plasma membrane to have the thickest and widest lipid membrane and causes the line on the graph to decrease at a slower rate. Conclusion - The length of TMD in the protein correlates with the width of the lipid transmembrane domain the type of amino acid, position of where it takes place and thickness of transmembrane Domain contributes to how the hydropathy plot results will turn out. The plasma membrane has the thickest and widest lipid membrane domain. References https://www.youtube.com/watch?v=d-N1c4AbgO8 https://www.youtube.com/watch?v=frVoetPCMWY https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/sec61 Chrome File Edit View History Bookmarks Profiles Tab Window Help 67% 11 Wed 3:15 AM omarreya : Quiz Sub x S Dashboard x C Chegg Sex Library Ge x PeopleSof x Search re х .. Abdullah х fos Free Onlin X ► YouTube х N Netflix x + . ben.desire2learn.com/d21/Ims/quizzing/user/quiz_submissions attempt.d2l?isprv=&qi=138689&ai=1166944&isinPopup=0&cfql=0&from QB=0&... ☆ Paused Update : Figure 4 PKA activity in the rat oviduct on day 1 of the estrous cycle following s.c. and intrabursal (i.b.) treatments with estradiol (E2) alone or with PLC inhibitor ET-18-OCH3, respectively. V, vehicle of drugs (s... and ib). All treatments were given 3-5 h before autopsy. Figures inside the bars indicate the number of replicas. Means with different letters are significantly different from each other (P
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