BIO302 Biochemical Messengers 2nd SEMESTER 2021/22 FINAL EXAMINATION
Hello, dear friend, you can consult us at any time if you have any questions, add WeChat: daixieit
BIO302
2nd SEMESTER 2021/22 FINAL EXAMINATION
Undergraduate – Year 4
Biochemical Messengers
TIME ALLOWED: 3 Hours
Section A
Question A1
During the emergence of neural crest cells from the neural tube, the expression of cadherins dynamically changes. Chick neural tube and neural crest cells were stained for N-cadherin (Ncad, red) as well as cadherin-6B (cad6B, green) (panel A) and cad6B (red) as well as cadherin-7 (cad7, green) (panel B). In the panel B, arrows indicate cells that have just detached from the neural tube and arrowhead shows the cells expressing both cadherins. Chick neural tubes were injected with adenoviruses expressing Ncad (panel C), cad7 (panel D), or mutant Ncad (cN390Δ, panel E), and the virus-infected cells were stained at 30 hours after the injection. Arrows indicate cells migrating along the dorsolateral pathway. Arrowheads point to infected cells localized in the roof plate and neural tube lumen. Explain these results (10 marks) and discuss the roles of cadherins for neural crest cell migration (15 marks).
Question A2
The Drosophila gene eyeless (ey) is homologous to the mouse Small eye (Pax-6) gene, and encodes a transcription factor. Panel A of the figure below indicates scanning electron micrograph of an ectopic eye (arrowhead) in the head region formed by the expression of the ey in antennal imaginal disc. Panel B is a higher magnification of the panel A and the ectopic eye is at the left. The photoreceptor clusters are shown in the ectopic eye (panel C) and the normal eye (panel D), respectively. Panel E indicates the scanning electron micrograph of an ectopic eye on a fruit fly leg induced by the expression of mouse Pax-6. Explain the mechanism for ey/Pax-6 to control animal eye formation (10 marks) and discuss how you can test whether the ectopic eyes are functional (15 marks).
Section B
Question B1
Sonic hedgehog (Shh) functions as the signal for anteroposterior patterning in the limb. Panel A of figure below indicates the expression of Shh in chick limb bud (black staining) with the anterior (A)-posterior (P) axis. Shh-expressing cells were implanted to the anterior (panel C) or posterior (panel D) margin of limb bud, and then the embryos were stained seven days after grafting. Panel B is an unimplanted control limb. The identities of digits (II, Ill, or IV) and long bones (H [humerus], R [radius], or U [ulna]) are shown. The anterior margins of limb buds were infected with the retrovirus to express Shh, and then the embryos were assayed for Hoxd-13 (one of homeotic genes) expression (arrows). Panels E and F indicate control limb bud and infected limb bud, respectively. Explain these results (10 marks) and discuss how Shh controls anteroposterior patterning in limbs (15 marks).
Question B2
Dorsoventral patterning in Drosophila embryo requires Decapentaplegic (Dpp) and Shortgastrulation (Sog). Using Xenopus embryo, Dpp mRNA was injected into each of four animal cells at the 8-cell stage, and some of the embryos were further injected with Sog mRNA into a vegetal blastopore. The embryos were then developed until stage 39 (panel A). Panel B indicates a single wild-type Xenopus embryo (top) and four embryos injected by Dpp mRNA (bottom). These embryos only contain ventral mesoderm. Panel C shows four embryos injected with both Dpp and Sog mRNAs. Explain these results (10 marks) and discuss the conservation as well as plasticity of dorsoventral patterning mechanisms during animal evolution (15 marks).
Section C
Question C1
Development of mammary glands occurs during puberty in a multiple-step process. One key step is the formation of terminal end buds (TEB) that will form the ductal tree as shown in the figure below.
TEB structures must proliferate and invade into the fat pad (matrix of adipocytes and connective tissue) for branch formation in the adult mammary glands. To understand the roles of metalloproteases (e.g. Mmp14) and integrins (Itgb1) for this process, the following results were obtained using Eph4 cells capable of branching/invading in a gel matrix.
In Figure 1 below, Eph4 cells were observed by microscopy in a gel matrix. The cells were treated with the Mmp14 inhibitor, GM6001, Mmp14-shRNA (short hairpin RNA) to attempt to silence Mmp14 gene expression, or a ctrl-shRNA containing a random sequence. The untreated cells are also shown (ctrl).
In Figure 2 below, the amounts of phosphorylated Erk (pErk) and total Erk (Erk) were quantified in (A) untreated (ctrl), GM6001, (B) ctrl-shRNA, Mmp14-shRNA, or (C) Itgb1-shRNA treated Eph4 cells by immunoblot. The bar graphs indicate the relative ratios of pErk to Erk (panels A-C).
A
B
C
In Figure 3 below, Eph4 cells lysates were first immunoprecipitated using control normal IgG (IgG) or anti-Mmp14 antibody (Mmp14) followed by immunoblot using anti-Mmp14 antibody (WB: anti-Mmp14) or anti-Itgb1 antibody (WB: anti-Itgb1). Total cell lysates were also directly analyzed by immunoblot (Cell lysate). The bands corresponding to Mmp14, IgG, and Itgb1 are indicated by arrows (panel A). In panel B, Eph4 cells expressing monomeric Cypet (Excitation: 435nm; Emission: 477nm) labelled FLAG tagged human Mmp14 (MMP14F mCypet) and monomeric Ypet (Excitation: 517nm; Emission: 530nm) labelled Itgb1 (ITGB1 mYpet) were subjected to a FRET assay.
Answer the following questions:
1) Based on the results of Figure 1, what can you conclude? (2 marks)
2) Are the results of Figure 2A and B the same or different? Explain your rationale. (4 marks)
3) What can you conclude based on the results of Figures 2B and 2C? (2 marks)
4) Is the effect of Mmp14 on branching/invasion of Eph4 cells mediated by activation of the ERK pathway? If your answer is yes, explain why. If no, explain what experiments might corroborate this assertion. (6 marks)
5) What is your conclusion from Figure 3A? (6 marks)
6) For the FRET experiment in Figure 3B, which wavelengths were used for the excitation as well as for recording the emission? (3 marks). The middle panel (ITGB1mYpet) shows areas with intense fluorescence (white arrow) that are absent in the right panel (FRET). How do you interpret this? (2 marks)
Question C2
Apelin receptors (APJ) are GPCRs that may be targeted to treat heart diseases.
In Figure 1 below, CHO cells were transfected with empty vector (Mock), hemagglutinin epitope (HA)-tagged APJ (HA-APJ), Myc epitope tagged (Myc)-APJ (Myc-APJ), or both (Co-transfection). The cell lysates from HA-APJ and Myc-APJ-transfected cells were also mixed prior to SDS-PAGE (Mix). Each cell lysate was first immunoprecipitated using anti-HA epitope antibody (IP HA) followed by immunoblot with anti-Myc epitope antibody (Myc) (upper panel). Each of the total cell lysates was also directly examined by immunoblot using anti-Myc epitope antibody or anti-HA epitope antibody (HA).
For BRET studies (Figure 2 below), CHO cells were transfected with APJ-Rluc and APJ-EGFP plasmids or mouse orexin type 2α receptor (mOX2αR)-Rluc and mOX2αR-EGFP plasmids. BRET saturation curves were obtained with a constant amount of donor-labeled protein with increasing amounts of acceptor-labeled protein. A luciferin was added for the BRET measurements.
In Figure 3 below, CHO cells were transfected with empty vector (Mock), a vector to overexpress APJ (APJ), control shRNA (shCTRL), or shRNA directed against APJ (shAPLN). Phosphorylated AKT (pAKT), total AKT (AKT), phosphorylated ERK (pERK), and total ERK (ERK) were quantified in the cell lysates by immunoblot (panels A and B). The bar graphs in panels C and D show the ratios of pAKT to AKT as well as pERK to ERK. Consider significant upregulation of AKT and ERK levels in the APJ-overexpressing cells (immunoblot A) and no changes for AKT and ERK levels in the shAPLN-expressing cells (immunoblot B)
Answer the following questions:
1) What can you conclude from Figure 1? (4 marks)
2) Explain why there is no band with the “Mix” lane in the upper panel of Figure 1? (6 marks)
3) Why was the rationale to perform the experiments in Figure 2? Is the result as expected (Yes/No)? (3 marks)
4) What was the purpose of having mOX2αR-Rluc and mOX2αR- EGFP in Figure 2? (6 marks)
5) What can you conclude from Figure 3? (6 marks)
Section D
Question D1
The balance between lipolysis and lipogenesis is controlled by Glucocorticoid receptor- (GR) and peroxisome proliferator-activated receptor- (PPAR), respectively. GR activation is achieved by serine phosphorylation at amino acids 212, 220, and 234. Protein phosphatase 5 (PP5) may regulate lipolysis and lipogenesis balance by an as-yet unknown mechanism. To better understand this mechanism, the following results were obtained. Mouse embryonic fibroblasts (MEFs) can be induced to differentiate into adipocytes by Dexamethasone (Dex).
Figure 1 below shows the results of immunoblotting to detect PP5, FLAG-tagged PP5 (FlagPP5), and HSP90 in MEFs derived from wild-type (WT) and PP5 knock-out (KO) embryos, as well as KO MEFs expressing FlagPP5 (KO-R).
Figure 2 below indicates Nile Red staining of undifferentiated MEFs (U) and adipocyte-induced MEFs (D) from WT and PP5 KO embryos as well as PP5 KO MEFs expressing FlagPP5 (PP5 Rescue).
Panel A in Figure 3 below shows the results of immunoblots to detect GR and PPAR in WT and PP5-KO MEFs. In panel B, WT and PP5-KO MEFs were treated with (+) or without (-) Dex, and then the cell lysates were analyzed to quantify GR phosphorylated at Ser 212 (pGR S212), Ser 220 (pGR S220), or Ser 234 (pGR S234) as well as total GR. The calculated ratios of phosphorylated GR to total GR are also plotted as bar graphs.
B
Figure 4 below indicate Nile Red staining of WT MEFs and PP5KO MEFs expressing either wild-type GR (WT-GR) or mutant GR in which Ser 203, 211, and 226 were all substituted with Ala (GR3A). The bar graph shows the amount of lipid in each cell type.
Answer the following questions:
1) In Figure 1, what was the purpose of expressing FlagPP5 in the KO cell line? (6 marks)
2) What can you conclude from the results of Figure 2? (4 marks)
3) What can you conclude from Figure 3A? (4 marks)
4) What do you conclude about the roles of PP5, according to Figure 3B? (4 marks)
5) What can you conclude from Figure 4? (7 marks)
Question D2
In prostate cancer, the roles of brain-derived neurotrophic factor/tropomyosin receptor kinase B (BDNF/TrkB) are not fully understood. To evaluate the potential importance of BDNF signaling pathway, four human prostate cancer cell lines, PC3, PC3M, DU145, and LNCaP (obtained from metastatic lesions) were analyzed in the following studies.
Figure 1, below, indicates the amounts of TrkB, BDNF, and β-Actin in DU145, PC3M, PC3, and LNCaP cell lysates by immunoblotting.
In Figure 2 below, four human prostate cancer cell lines were seeded and allowed to migrate (Migration)/invade (Invasion) for 24 hours in culture medium containing no additive (Control), BDNF, or BDNF and the TrkB inhibitor (BDNF+K252a). The bar graphs show the relative migration and invasion rates of each cell line under three different conditions.
Figure 3 below shows the cellular morphology of PC3 and LNCaP cells transfected with an empty vector (Vector) or vector to drive expression of TrkB (top panel). In the bottom panel, three epithelial-mesenchymal transition (EMT) markers, Vimentin, Twist, and Snail were quantified in cell extracts by immunoblot. The numbers below the bands indicate the calculated fold-changes in TrkB relative to Vector.
Figure 4 (below) indicates the relative migration (left) and invasion (right) rates of PC3 cells transfected with an empty vector (Vector) or vector that expresses TrkB, alongside the addition of BDNF (BDNF) or no-addition (Control).
Figure 5 (below) shows the amounts of TrkB and β-Actin in DU145 cells stably expressing a non-targeting sequence shRNA (shRNA-NT) or TrkB-specific shRNA (TrkB-shRNA- 1) by immunoblot (left panel). Vimentin, Twist, Snail, and β-Actin levels were also examined (middle panel). The right panel indicates the relative number of cells undergoing migration or invasion in the two cell treatment conditions.
Figure 6 (below) indicates the amounts of phosphorylated ERK (p-ERK), phosphorylated AKT (p-AKT), and β-Actin in PC3 and DU145 cells in the absence (-) or presence (+) of BDNF or K252a. The bar graphs show the fold change of pERK or p-AKT compared to β-Actin under the different conditions.
Answer the following questions:
1) What can you conclude from Figure 1? (3 marks)
2) Explain the effects of BDNF and BDNF+K252a on the different cell lines shown in Figure 2. How would you confirm that K252a is the specific inhibitor? Briefly explain whether delivering K252a to a prostate cancer patient (e.g. through intravenous injection) could be the valid therapy. (4 marks)
3) Why is it important to analyze the levels of EMT markers in Figure 3? (3 marks)
4) Briefly explain the differences between migration and invasion assays. What are the effects of BDNF and TrkB on migration and invasion of PC3 cells (Figure 4)? In a prostate cancer patient, which cells would synthesize BDNF activating TrkB on the prostate cancer cells? What type of signaling is this (e.g. endocrine, synaptic, long distance, etc)? (4 marks)
5) What conclusions can be made from the data in Figure 5 (left and middle panels)? Which characteristics do you analyze to better understand the phenotypes of DU145 cells by TrkB-shRNA- 1 treatment? (4 marks)
6) What can you conclude from Figure 5 (right panel)? How would you confirm this effect is specific, and not an experimental artifact? In Figure 6, why the immunoblot for p-ERK shows two bands? Which data are missing to interpret the results of immunoblots for p-ERK and p-AKT? (4 marks)
7) What can you conclude from Figure 6? (3 marks)
2023-05-07