User:Z3375490

From CellBiology

--Z3375490 (talk) 15:22, 22 May 2014 (EST)

Lab 9

Hypothesis

The normal mechanism through which cell death occurs in the given is apoptosis

Aims

We will measure apoptotic rate by subjecting the tissue culture to Flow cytometry, DNA marker, Hoecht staining and FLICA..

Key Techniques & Procedures

Flow cytometry is used to detect the fragmented DNA.

Conofocal microscopy to assesses the morphological features of apoptosis such as apoptotic blebs.

DNA marker to detect the DNA fragmentation. The fragmented DNA, from the apoptic cell, will present a DNA ladder in comparison to the unfragmented live cell.

Hoecht staining of apoptotic nuclei (with Hoescht 33342 as a blue stain) to determine the condensation and fragmentation of the nuclei. Hoechst 33342 binds preferentially to adenine-thymine (A-T) regions of DNA. This stain binds into the minor groove of DNA and exhibits distinct fluorescence emission spectra that are dependent on dye:base pair ratios.

FLICA (flourochrome inhibitor of caspase) is a simple yet accurate method to measure apoptosis via caspase activity in whole cells. It applies the green fluorescent inhibitor probe FAM-VAD-FMK to label active caspase enzymes in the cell samples. FLICA probes are comprised of an inhibitor peptide sequence that binds to active caspase enzymes, a fluoromethyl ketone (FMK) moiety that facilitates an irreversible caspase binding event, and a fluorescent tag (either carboxyfluorescein or sulforhodamine B) reporter. The green poly caspase inhibitor FAM-VAD-FMK utilizes the fluorophore carboxyfluorescein and the multi-enzyme recognition sequence valine-alanine-aspartic acid (VAD). Upon encountering active caspases, the FLICA probe interacts with the enzymatic reactive center of the enzyme via the peptide recognition sequence, forming a covalent thioether adduct with the enzyme through the FMK moiety. After a simple wash step, the remaining green fluorescent signal is a direct measure of caspase activity at the time the probe was added.

Suppliers

Thermo Scientific Pierce Hoechst 33342 Fluorescent Stain is a high-quality solution of Hoechst dye for fixed- and live-cell fluorescent staining of DNA and nuclei in cellular imaging techniques Thermo Scientific Pierce Hoechst 33342 Fluorescent Stain

FLICA™, Fluorescent-Labeled Inhibitor of Caspases, . FLICA-FAM

The E-Gel® Low Range Quantitative DNA Ladder with ethidium bromide staining. The E-Gel® Low Range Quantitative DNA Ladder

Flow Diagram

Cell sample (non-adherent cells) → add necessary fluorescent dye (FLICA™)→ perform flow cytometry → with blebs present, apoptosis is proven to have occurred

For Hoescht 33324:

Wash sample with DBPS -> add Hoescht 33342 Working Solution to incubate for 5-10 minutes in the dark-> wash off excess Hoescht 3342 ->mount sample & detect flouresence

DNA assay: add 50ul of cell smaple to cuvette -> add 1.5 ml Hoescht 33342 to each cuvette -> cover and incubate for 10 mintues -> measure florescence ->prepare standard curve

Outcomes

If apoptotic blebs and DNA fragmentation are detected by flow cytometry, the results suggest that the cells died by apoptosis. The Hoechst stain will present the apoptotic nuclei with a blue stained DNA. The FLICA will present a green stain of the caspase that are involved with apoptosis i.e. Caspases 3, 8, 9. The E-gel will present fragmented DNA, from the apoptic cell, will present a DNA ladder in comparison to the unfragmented live cell.

--Z3375490 (talk) 17:04, 15 May 2014 (EST)


Peer Review

Group 2 peer review – from cytoplasm to mitochondria

So far the project is looking nice and organized. However, here are a few tips:

Could you please add the Group projects hyperlinks that directly navigate to each project page i.e." UNSW ANAT3231 Course Coordinator Dr Mark Hill
2014 Projects: Group 1 | Group 2 | Group 3 | Group 4" at the top of the page.

For the introduction, it would be best to split this section into ‘intro’, ‘structure’ ‘function’ and ‘recent research/discovery’ because the last half talks about the recently discovered processes.

It might be good to talk about the urea cycle (which is pyruvate is apart of), why protein is needed in the mitochondria (i.e. breakdown), - Add more pictures (acts least 1 picture to each section). Also maybe draw a simple flow diagram of the TOM complex. - Mention ATP synthase. Including the motor diagram

It is good that you mentioned the protein transport, however it might also be essential to include the transport of ATP, i.e. respiratory chain and oxidative phosphorylation. Describe the origin of pyruvate. And include diagrams/processes that occur specifically in the mitochondrial membrane

Some sections seem to be missing i.e. Rna + lipd transport into mitochondria. If this transport process is too complex, it is best to ask Mark if it is needed or not in the project.

The disease and research part seems fine but may require more detail and pictures.

Sorry, I feel as though am going of a tangent with this and that I might be describing process that occur in the matrix. Please ask Mark if these are essential at all for the project.

However, the referencing looks great and use of image in most sections are nice and orgainised.


Group 3 peer review - cytoplasm to nucleaus transport

The intro, history, the nuclear import pathways and Future research needs to be completed. The diseases section requires more information and images.

For the NPC models although all 4 are accepted, which 1 is the most accepted? Also included images of each individual model.

The RanGTP/GDP and receptors section seems fine and informative. Create a separate glossary sections like Mark does for the lectures.

Please review you subtitling because some I am not sure if some of the subtitles are meant to be main titles or empty subsection i.e. ‘Nuclear Ionisation sequence’.

The referencing looks great.


Group 4 peer-review – neuron some to processes

Many sections are missing. i.e. mechanisms, dendric transport, diseases, or need more detail.

Please include more images (at least one for each section) sine there is only 1 photo.

Many of the headings are not enlarged or in bold such as "different types of nuerons"

There is a citation error in your referencing please fix this.

The fast and slow transport model is difficult to understand.

Nice table by the way on motor proteins. I also liked the description of the 2 model on kinesin movement. However it would be great if there were images to complement these models.

In addition, the intro does not describe the neuron processes. Please provide a brief description about this structure.

It would be great if there was a glossary of terms and abbreviation.

Sorry I cannot provide a good enough review because this project is really incomplete.

However, the referencing looks really good and the description of Motor proteins is the best part of the project.

Thanks and good luck.


cytoskeleton excercise

Graph of cell.png [[1]]


--Z3375490 (talk) 15:15, 17 April 2014 (EST)

--Z3375490 (talk) 15:11, 10 April 2014 (EST)

Immunochemistry H/W

-Identify an antibody that can been used in your group's transport project.

MOUSE ANTI CLATHRIN HEAVY CHAIN - a monoclonal antibody.

Used in immunoprecipitation, western blotting, Immunofluorescence, ELISA

datasheet


-Identify the species deriving the antibody.

Raised from mice.

(Reacts with pigs, humans, bovine and rats.)


-Identify the working concentration for the antibody.

IgG concentration 1.0mg/ml

--Mark Hill (talk) 14:29, 1 May 2014 (EST) This is the concentration of the antibody as it arrives to the user, not the working concentration. That is the final dilution used experimentally. It is not shown on the data sheet so you needed to look at one of the references.

-Identify a paper that has used this antibody.

L Macůrek, M Zíková, E Dráberová, P Dráber Monoclonal antibody BF-06 against the heavy chain of clathrin. Folia Biol. (Praha): 2003, 49(6);238-40 PubMed 14748440

| PubMed

Immunochemistry Lab

- Identify an antibody against an adhesion junction protein that is commercially available.

- Add a link to the original data sheet page and identify the type of adhesion junction.

- Include the following information: type of antibody (polyclonal, monoclonal), species raised in, species reacts against, types of application uses, and if available any reference using that antibody.

MOUSE ANTI HUMAN CD324

MOUSE ANTI HUMAN CD324 Datasheet

Type: Cadherin

The mouse anti-human CD234 is a monoclonal antibody raised in mice and reacts against humans. It used in Western bolt and frozen immunohistochemistry.

References: U H Frixen, J Behrens, M Sachs, G Eberle, B Voss, A Warda, D Löchner, W Birchmeier E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells. J. Cell Biol.: 1991, 113(1);173-85 PubMed 2007622

| Pubmed

Helmut Bühler, Gerhard Schaller Transfection of keratin 18 gene in human breast cancer cells causes induction of adhesion proteins and dramatic regression of malignancy in vitro and in vivo. Mol. Cancer Res.: 2005, 3(7);365-71 PubMed 16046547

| Pubmed

Alexis Chassignol, Emmanuelle Brasnu, Christophe Baudouin, Luisa Riancho, Jean-Michel Warnet, Françoise Brignole-Baudouin In vitro interactions between peripheral blood lymphocytes and the Wong-Kilbourne derivative of Chang conjunctival cells. Invest. Ophthalmol. Vis. Sci.: 2012, 53(3);1492-8 PubMed 22328639

| Pubmed


--Z3375490 (talk) 15:22, 3 April 2014 (EST)



Lab 4 work - week 5

--Mark Hill (talk) 14:33, 1 May 2014 (EST) You have identified 5 papers that relate to your group topic, your descriptions are brief but OK. Simply restating the paper title and part of the abstract would not be acceptable within the project.

Phagocytosis

Contemporaneous cell spreading and phagocytosis: Magneto-resistive real-time monitoring of membrane competing processes

The article ‘Contemporaneous cell spreading and phagocytosis: Magneto-resistive real-time monitoring of membrane competing processes’ by Shoshi et al investigate that during phagocytosis the cell membrane expands, to engulf particles (in this case beads on surfaces), by cell spreading. The engulfment rate was additionally measured using real-time magneto-resistive monitoring, with an average of 3 beads per minute. Correspondingly, the rate of engulfment was not a linear function but is high at an early stage, then decreases steadily until saturation.

Reference

A Shoshi, J Schotter, P Schroeder, M Milnera, P Ertl, R Heer, G Reiss, H Brueckl Contemporaneous cell spreading and phagocytosis: magneto-resistive real-time monitoring of membrane competing processes. Biosens Bioelectron: 2013, 40(1);82-8 PubMed 22770907

| Pubmed

Copyright

@2012 Shoshi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: '‘Contemporaneous cell spreading and phagocytosis: Magneto-resistive real-time monitoring of membrane competing processes’ A. Shoshi, J.Schotter, P.Schroeder, M.Milnera, P.Ertl, R.Heer, G.Reiss, H.Brueckl (2012) Biosensors andBioelectronics40(2013)82–88. doi: 10.1016/j.bios.2012.06.028. Epub 2012 Jun 23.


Rab35 Mediates Transport of Cdc42 and Rac1 to the Plasma Membrane during Phagocytosis

‘Rab35 Mediates Transport of Cdc42 and Rac1 to the Plasma Membrane during Phagocytosis’ journal, by Shim et al, put forward that a Rab GTPases, called Rab35, regulate trafficking from the cell membrane to the cytoplasm. Rab35 is a specific regulator of the actin cytoskeleton in the plasma membrane and play a role in filopodia and lamellipodia. In addition, Rab35 is essential for Cdc42 and Rac1 localization at an activated plasma membrane, when in contact with foreign particles and therefore is crucial for actin rearrangement during phagocytosis. Drosophilas were used to demonstrate the hypothesis and presence/absence of Rab35 effecting phagocytosis.

Reference

Jaewon Shim, Sun-Min Lee, Myeong Sup Lee, Joonsun Yoon, Hee-Seok Kweon, Young-Joon Kim Rab35 mediates transport of Cdc42 and Rac1 to the plasma membrane during phagocytosis. Mol. Cell. Biol.: 2010, 30(6);1421-33 PubMed 20065041

| Pubmed

Copyright

@2010 Shim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: ‘Rab35 Mediates Transport of Cdc42 and Rac1 to the Plasma Membrane during Phagocytosis' Jaewon Shim, Sun-Min Lee, Myeong Sup Lee, Joonsun Yoon, Hee-Seok Kweon, and Young-Joon Kim (2010) Mol Cell Biol. 2010 Mar;30(6):1421-33. doi: 10.1128/MCB.01463-09. Epub 2010 Jan 11.


Plasma membrane tension orchestrates membrane trafficking, cytoskeletal remodeling, and biochemical signaling during phagocytosis

The article ‘Plasma membrane tension orchestrates membrane trafficking, cytoskeletal remodeling, and biochemical signaling during phagocytosis’ focus on the 2 phases of pseudopod extension that included actin polymerization pushing the membrane forward and increased membrane tension using high-resolution microscopy of macrophages attempting to internalize an IgG-opsonized glass surface. A 50% increase in tether force was observed in phagocytic cells, compared to the resting cells membrane tension. Additionally, inward bead movement (engulfment) and ingestion is most probably due to contraction and exocytosis activation. This confirms that membrane tension is an exocytosis activator and that exocytosis is required for phagocytosis to complete.

Reference

Thomas A Masters, Bruno Pontes, Virgile Viasnoff, You Li, Nils C Gauthier Plasma membrane tension orchestrates membrane trafficking, cytoskeletal remodeling, and biochemical signaling during phagocytosis. Proc. Natl. Acad. Sci. U.S.A.: 2013, 110(29);11875-80 PubMed 23821745

| Pubmed

Copyright

@2013 Masters et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: ‘Plasma membrane tension orchestrates membrane trafficking, cytoskeletal remodeling, and biochemical signaling during phagocytosis' Thomas A. Masters, Bruno Pontes, Virgile Viasnoffa, You Li, and Nils . Gauthier, (2013) Proc Natl Acad Sci U S A. 2013 Jul 16;110(29):11875-80. doi: 10.1073/pnas.1301766110. Epub 2013 Jul 2.


Circular Dorsal Ruffles

Integrins traffic rapidly via circular dorsal ruffles and macropinocytosis during stimulated cell migration

The article ‘Integrins traffic rapidly via circular dorsal ruffles and macropinocytosis during stimulated cell migration’ presents that upon cell stimulation with platelet derived growth factor do not undergo significant endocytosis at ventral focal adhesions. Instead, it is redistributed to dorsal circular ruffles. This was assessed using 4-D confocal live-cell imaging. Additionally, integrins transit through recycling endosomal compartments to repopulate new focal adhesion on the ventral surface.

Reference

Zhizhan Gu, Erika H Noss, Victor W Hsu, Michael B Brenner Integrins traffic rapidly via circular dorsal ruffles and macropinocytosis during stimulated cell migration. J. Cell Biol.: 2011, 193(1);61-70 PubMed 21464228

| Pubmed

Copyright

@2013 Zhizhan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: ‘Integrins traffic rapidly via circular dorsal ruffles and macropinocytosis during stimulated cell migration' Zhizhan Gu, Erika H. Noss, Victor W. Hsu, and Michael B. Brenner, (2011) The Journal of Cell Biology 2011 Apr 4;193(1):61-70. doi: 10.1083/jcb.201007003


Image

[Macrophage pseudopod extension occurs in two phases during FcγR-mediated frustrated phagocytosis and correlates with membrane and cytoskeleton dynamics]

Pnas.1301766110fig01.jpg

Localization of human nuclear envelope proteins in Drosophila.

Polytene nuclei from salivary glands (top row z-series; middle row section) and diploid cells from imaginal discs (bottom row) were obtained from transgenic stocks and stained with antibodies specific for the human nuclear envelope proteins. All of the human proteins localized to the Drosophila nuclear envelope, with Lamin B2 showing aggregation.

Reference

Sandra R Schulze, Beatrice Curio-Penny, Sean Speese, George Dialynas, Diane E Cryderman, Caitrin W McDonough, Demet Nalbant, Melissa Petersen, Vivian Budnik, Pamela K Geyer, Lori L Wallrath A comparative study of Drosophila and human A-type lamins. PLoS ONE: 2009, 4(10);e7564 PubMed 19855837

| PLoS ONE

Copyright

@2009 Schulze et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: Sandra R. Schulze, Beatrice Curio-Penny, Sean Speese, George Dialynas, Diane E. Cryderman, Caitrin W. McDonough, Demet Nalbant, Melissa Petersen,Vivian Budnik,Pamela K. Geyer, Lori L. Wallrath 'A comparative study of Drosophila and human A-type lamins' PLoS ONE(2009) 4 (10):e7564. doi: 10.1371/journal.pone.0007564.


Note - This image was originally uploaded as part of a student project and may contain inaccuracies in either description or acknowledgements. Please contact the site coordinator if the uploaded content does not meet the original copyright permission or requirements, for immediate removal.

--Z3375490 (talk) 15:11, 27 March 2014 (EST)



Homework week 4

Picture task

Localization of human nuclear envelope proteins in Drosophila.png

[Localization of human nuclear envelope proteins in Drosophila]

Localization of human nuclear envelope proteins in Drosophila.

Polytene nuclei from salivary glands (top row z-series; middle row section) and diploid cells from imaginal discs (bottom row) were obtained from transgenic stocks and stained with antibodies specific for the human nuclear envelope proteins. All of the human proteins localized to the Drosophila nuclear envelope, with Lamin B2 showing aggregation.

Reference

Sandra R Schulze, Beatrice Curio-Penny, Sean Speese, George Dialynas, Diane E Cryderman, Caitrin W McDonough, Demet Nalbant, Melissa Petersen, Vivian Budnik, Pamela K Geyer, Lori L Wallrath A comparative study of Drosophila and human A-type lamins. PLoS ONE: 2009, 4(10);e7564 PubMed 19855837

| PLoS ONE

Copyright

@2009 Schulze et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: Sandra R. Schulze, Beatrice Curio-Penny, Sean Speese, George Dialynas, Diane E. Cryderman, Caitrin W. McDonough, Demet Nalbant, Melissa Petersen,Vivian Budnik,Pamela K. Geyer, Lori L. Wallrath 'A comparative study of Drosophila and human A-type lamins' PLoS ONE(2009) 4 (10):e7564. doi: 10.1371/journal.pone.0007564.


Identify a recent research article (not review) that uses either confocal microscopy or super-resolution microscopy as one of the study's techniques. Explain briefly (1 paragraph) how the microscopy technique specifically contributed to the article's findings.

The article ‘In Vitro Whole Blood Clot Lysis for Fibrinolytic Activity Study Using D-Dimer and Confocal Microscopy’ used confocal microscopy as the study technique to validate the in vitro WB (Whole Blood) clot lysis method for fibrinolysis (fibrinolytic activity). Protocols and staining procedures were undertaken to assess the WB clot morphology such as merocyanine 540 flourescent dye (MC-540) for labeling the red blood cells in red and Alexa Fluor 488 human fibrinogen conjugates (F-13191) for fibrin fibers in green. Before the WB clot was examined, by the confocal microscope, an antifade fluorescence mounting medium was added. The conofocal microscope assisted in evaluating the in virto WB clot lysis through the assessement of fibrinolytic activity. This is seen in showing that WB clot that was treated with SK (strepyokinase) and PPP (platelet poor plasma), separately, had fibrin fibers that became thinner with increased branching and separated or disappeared from the RBCs over time or by increased SK. The confocal microscope also showed that fibrin was thicker in PPP than in SK treated clot.

Pubmed

Reference

Abuzar Elnager, Wan Zaidah Abdullah, Rosline Hassan, Zamzuri Idris, Nadiah Wan Arfah, S A Sulaiman, Zulkifli Mustafa In vitro whole blood clot lysis for fibrinolytic activity study using d-dimer and confocal microscopy. Adv Hematol: 2014, 2014;814684 PubMed 24660000

| Pubmed

Copyright

@2014 Elnager et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: 'In Vitro Whole Blood Clot Lysis for Fibrinolytic Activity Study Using D-Dimer and Confocal Microscopy' Abuzar Elnager, Wan Zaidah Abdullah, Rosline Hassan, Zamzuri Idris, Nadiah Wan Arfah, S. A. Sulaiman, and Zulkifli Mustafa (2014) Adv Hematol. 2014;2014:814684. doi: 10.1155/2014/814684. Epub 2014 Feb 11.



--Z3375490 (talk) 15:09, 20 March 2014 (EST) http://ajpcell.physiology.org/content/284/4/C1083.full-text.pdf+html

12661552

--Z3375490 (talk) 15:47, 13 March 2014 (EST) Hello, Everybody. Buongiorno! Mi piace Cell Biology.

Lab 1

Add your own student page to the site. X 
Add your signature for Lab attendance. X - click signature tab
Add a sub-heading. X
Add an external Link. PubMed X
Add an internal Link. [Lab 1]

[model for lipid modulation]

File:The two stage model for lipid modulation of the enzyme activity.png The two stage model for lipid modulation of the enzyme activity

[1]

Immunohistochemical detection of DNase II.png

--Mark Hill (talk) 14:16, 3 April 2014 (EST)

  • Your internal link to an image that you do not want displayed should look like this model for lipid modulation.
  • Also if you remove the space from the beginning of each line the text will format correctly.
  • Your reference tag is incorrectly formatted, that is why you are getting red text. Should look like this <ref><pubmed>24603867</pubmed></ref> (without the nowiki tags)
  • For lab 2 task the image should appear on this current page rather than be linked from here. As I show below.
Localization of human nuclear envelope proteins in Drosophila.png
  1. Ziga Zebec, Andrea Manica, Jing Zhang, Malcolm F White, Christa Schleper CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus. Nucleic Acids Res.: 2014, 42(8);5280-8 PubMed 24603867