User:Z3333208

From CellBiology

Group 7 - G protein

Lab 12

1) Identify a current technique used in gene sequencing.

Next Generation Gene Sequencing


2) Identify a recent cell biology research paper that has used microarray technology.

<pubmed>22629434</pubmed>


3) What aspect of the research findings were contributed by the microarray technique.

Mice that lack EphA4 are able to regenerate better following spinal cord injury. This is because EphA4 is an axon guidance molecule which inhibits axonal regeneration in many directions. To determine how EphA4 acts on the axons following injury, a microarray was done on spinal cord tissue from both mice with spinal cord injury (hemisection of cord) and mice with sham injury (laminectomy). Two types of mice were used; wild type and EphA4 knockout mice. RNA from the four groups was collected and purified and hybrisised to Affymetrix All-Exon Array 1.0 GeneChips. Several studies showed that several pathways are altered in the EphA4 knockout mice. In particular the analysis indicated that a number of inflammatory genes (such as Arginase 1) showed increased expression in the wild type mice [1].

Therefore, we can speculate that EphA4 increases the inflammatory response following neural injury, hindering axonal regeneration.

Lab 10

  • Stem cell transplantation: SC/Brain, skin, bone marrow,skeletal m, liver, heart.
  • Most successful stem cell therapy? Bone marrow transplant.

Why muscle stem cell therapy?Italic text

  • No. of congenital diseases in muscle (muscular dystrophy)->no strategy for producing cure.
  • Muscle injury-> repair
  • Muscle wasting illnesses.

Problems with stem cellsItalic text Injecting stem cells will compete with endogenous stem cells (biggest impediment). Immunosuppression Failure to migrate Failure to enter stem cell niche Low engraftment (SC from one niche to target another)

How to eliminate competition from endogenous cells? Get rid of cells How to protect from immune system? Immunosuppress the system How to enhance migration? Damaging tissue, disrupts cell structure, and hence makes environment more favourable to migrate.

  • Chemotherapy, kills rapidly dividing cells very effectively.
  • Skeletal muscle has a great ability to regenerate.
  • BCNU- methylated chemotherapy.
  • Alkylating chemo: add methyl groups to DNA of all cells being exposed to the radiation.
  • If cells divide quickly they die, O6BG inhibits MGMT, so cells die-> e.g. Skeletal SC's
  • Normal cells would divide more slowly, there is a mechanism (MGMT) which removes methyl groups without killing cell
  • Cells that express MGMT (via a mutation) will be resistant to chemotherapy. Chemo resistant genes
  • This selective donor engraftment strategy is being done on bone marrow.
  • How to test the strategy: Mix mutated chemo resistant SC's & normal stem cells, radiate, observe the cells that survive.
  • Univeral donor- all sc from the mouse will be chemo-resistant.
  • Test resistance: Aus Tiger snake venom (notexin)-> dissolves all skeletal m, and this acticvates the SC in regenerating muscle (central nuclei)-> Add chem, if the cells are chemo resitence-> cells should survive, and muscle should regenerate a muscle bed.
  • SC of muscle are statelite cells.
  • Where do i isolate the muscle stem cells? Remove muscle, cut it up, enzymatically injury muscle, activates satelite cell (cell is no longer quiescent) which can then be retrieved. Statelite cells express CD34, attach them using antibodies then isolate cells using magnet.
  • Male donor, female recipient.
  • Biggest differnce is Genger- XY Chromosome v XX Chromosome, can be used to measure the amount of muscle bed produced by each type of SC. PCR can be used to measure the amount of Y chromosome in the muscle bed.


  • 15-20% of muscle nuclei are from donor. Presence of chemo-resitance donor sort of RECRUIT makes the endogenous cell regenerate the most of muscle bed.
  • Where did the other endogenous SC come from? Possibly nearby niches, by not eliminating every stem cell in a bed, these could come out and repopulate the area.
  • Improve: More effective killing of competition, and better protection of donor stem cells.

Lab 9

--Mark Hill 12:59, 17 May 2012 (EST) You have made a good critical assessment of each project including detailed sub-heading coverage. You may consider in future separating your assessment into pro/con or similar for the group to then more easily identify strengths and weaknesses.

Peer assessment

Group 1

  • Intro seems disorganised, you are trying to give away too much. formatting needs to suit the section and i don't think a table should go into intro. properties should be in another section. i like the inclusion of the image of the molecule.
  • History table is great, its colourful and content is really well researched.
  • Biosynthesis, a very detailed table, maybe instead of such a complex image you can make a student drawn flow chart of the steps and place that next to the table or within the table as another column.
  • Regulation is very simplified, may you should elaborate some more here, add an image or graph of the differences with testosterone high vs low.
  • Signalling pathway has been done very well. its clear with subheadings, bullet points, and images are eye catching.
  • Normal function appears like a big slab of text which i have no intention of reading, you need to make people what to read it, add bullet points, images like the other sections of this project.
  • Abnormal function also well done but requires some images or something to break up all the text, but good use of subheadings.
  • Clinical uses table is very comprehensive, and seems easy to read and understand.
  • I do have issues with this section, i really don't understand what you are saying, what research you are talking about when you say "In a recent study, both injection and oral testosterone were compared to demonstrate their differing effects." etc. i don't understand the numbering (what are these sentences relating to?) when you say this study was done... you should post the reference for the study either with the paragraph or as a citation. Its very confusing, i don't know how many articles you are referring to.

Group 2

  • Image at the top of page, you should add a caption so people know what they are looking at.
  • Intro and history are good, maybe some more information can be added to intro so its more of a paragraph of what you will be discussing in the project.
  • Normal function, is very incomplete. needs more information, it a uniform structure- what i mean by that is, if you want to write in short paragraphs then do so for all subheadings don't do paragraphs and a basic hormone list.
  • Signalling pathway: is also incomplete. i like the table, its simple short and succinct. i think its good to say the basics in the table then further elaborate below, smart thinking.
  • Abnormal function section is excellent. it is simple, short sentences (so easily read), images (engaging), nice layout. great work. however, only one image is allowed to be used from wikipedia, i believe you have two in that table.
  • Therapeutic application is a good section, it has diagrams, its simply worded, short sentences, interesting info. good work but i think you should add some more examples of other illnesses.


Group 3

  • A really great introduction. it is clear and well said, and does what an intro should do, that is introduce what you will be discussing, GREAT WORK. Same said for History
  • I don't really like how you listed 15 proteins but then only spoke about one. It seems confusing and if you had made a little table with the most well understood proteins, their structure, and function as headings i would of been more comprehensive, and would demonstrate a broader understanding of the proteins involved in signalling. I think anyone can list the proteins and say their involved!
  • Function section is lacking a lot. it has NO referencing so how are you meant to fill the referencing section before this project is due ?!? Its just a slab of text, it definitely needs subheadings, bullet points, images, diagrams, it needs to engage the audience and to be honest i didn't want to read past the second paragraph.
  • Current research: i don't think articles from 2002/07 are current to be blunt. i do like how you have a paragraph introducing the research though.
  • There is one image on the entire page and nothing really special about this project, its quite boring and to make it interesting you really should look at cell death lecture 2, because Mark spent a lot of time on this topic, you should use some of his images and you should definitely look at how he discussed the pathway, (using bold headings for steps and short sentences below each). I think you have a lot of work to do. Start your referencing soon because if you loose where your references then you would be plagiarising. Goodluck

Group 4

  • There is no large title saying NOTCH signalling- it should be added so people know what the page is about before reading.
  • Intro is short, thats fine so long as you expand on what is said throughout the project. History is also short, i heard this signalling pathways is fairly recent and difficult to study, but maybe just a few more points will make it more comprehensive- it seems like its all based on Morgan and the fly. is there nothing else about the signalling or abnormal diseases which relate to notch signalling that can be added to your table.
  • Image of Notch Signaling Network , has no copyright info attached!
  • surely there is more known about the Protein and receptor section. I understand you don't want too much information that people don't want to read, but i think your page is on the other side of the spectrum, with not enough information in all sections (apart from normal function- which is detailed, but still easily read, contains a picture, would be nice to see a few more)
  • Videos are a good addition but i don't think they make up for the lack of effort in most sections.
  • Futher research; seems like someone just pasted a few pubmed no. in this section. at least write a sentence or two about what the article is hoping to discover etc.

Group 5

  • Clever, creative intro.
  • History- great, nice detail, looks like someone put a lot of effort into it.
  • Mechanism of action, is another great section. info is in short and simple sentence layout (following Marks lecture material) i think its smart and clever thinking- you don't want to further elaborate i think the about of info is enough. nice picture, wish it could have more colour to it though- make it more appealing. MOVIE is a great idea- but very detailed like you warned
  • Disease table is also good, i like how you have little bit of information under each column makes it easy to read and understand. Dont know what has happened with ur references in this section.
  • This table under Key players in Wnt/β-catenin Signalling is nice work, will be good source of info when complete
  • Embryonic dev. is another clever interesting section you have added, i was interested in reading it.


Group 6

  • I don't think intro should have so many subheadings when under each there isn't much info, or many subheading size it too large?! But content is good, easy to read, simple nice work.
  • Structure of insulin section- it has a nice big photo which is eye catching but i have a few issues with it- NO copyright or info in regards to where photo came from. Nothing to say what green and purple is representing, no text to accompany image so i really don't know or believe what I'm looking at. Is purple insulin or the green or both? Its confusing. The specifications are good but surely there is more to say about this molecule.
  • History is poor, for example "1988 Discovery of PI3K" i don't understand what this even means!, i don't see a reference for me to look into it. seems like this section was done in ten mins, i think if you spent more time you could have a great intro i think insulin is so well known that there would be multiple reviews about its history and it just takes some initiative.
  • Insulin receptor: is good, clear, succinct. But i really don't think the image needs to be so massive..
  • WOW signalling pathway section is really really good, i actually read the info, theres not too much that its a drag to read and there is an image that relates. its a perfect balance of information to image.
  • Normal function section is good, has potential to be better by adding some figures, images, graphs. EG, pancreas islet cell image from histology slides from HISTOLOGY, figures of levels of insulin after food before, such things found in many books...
  • Abnormal section has good info but nothing to make me what to read it, think of adding some diagrams or graphs or something to engage your audience

Group 8

  • Intro is short and sweet, good. Pathway section is very nice, only suggestion is enlarge the image so people can see it without having to click
  • A couple of sections are untouched this is a bit disappointing because overall information on the page seems of a high standard.
  • The protein section is really good information, it would be nice to see some more images, possibly of the actually proteins interacting with molecules discussed.
  • Minor typo- Diagram of the adhesion molecule variant of the selecting group
  • Both normal function and abnormal function sections seem to have good information, but it isn't very eye-catching, i don't think people will want to read it. I think you might need to add a few images to these sections so its not just slabs of info, but good to see headings and subheadings.
  • Big issue with normal function section- NO REFERENCES? Definitely would need it, i don't think thats all your own work


Group 9

  • A nice succinct introduction, sounds scientific and provides I think a lot of information about p53 in a few paragraphs- which can then be elaborated on within the project. I can suggest adding a digram which can link into the topics discussed like the cell cycle or p53 molecule itself- in order to make it more attractive and eye catching.
  • Pathway section: I think having the text just as a slab of information (which is obviously the starting point of the work) is not very appealing to read. I can suggest you use some bullet points, numbering of pathways, bolding words... The information seems to be of average standard, i would assume this is a difficult pathway but i personally would want to see more information (as an example in the cell death lecture 2, Mark talks about the two pathways for apoptosis, the level of detail he uses is what i think you would need to use in this section- given how long we have had to work on the assignment).
  • Empty sections with nothing at all written is very sad and the only thing i suggest is ADD SOMETHING!
  • Obviously you need to work on the remaining sections which seem incomplete
  • Normal function section appears like information is really of good standard, but definitely needs images or something to make people want to read the entire section.
  • History section, looks eye catching, short succinct sentences, all referenced, gives great basic knowledge of the history- 10/10
  • Very good referencing throughout the page of the sections done, but particularly the history section!


Notes

Fungal- filamentous expansion throughout the media (yeast is common also- floating ontop of media)

bacterial- lots of small cells In contaminated media, a white milky material precipitate will be on base, or black spotted media. Decontaminate media before opening media (this would produce an aersol that can spread in area)- by bleach before discarding. Deal with dirty material last. quarantine new cell lines, grow separately for a few weeks so you dont introduce new contamination into lab.

Mycoplasma- smallest bacteria- live inside cell (cytoplasmic). Difficult to see- so routinely screen plates for mycoplasma (every couple months) PCR analysis or DNA screen for mycoplasms to check

  1. Neuronal cells - High glucose requirement, highly aerobic (will not survive low glucose)
  2. Non-neuronal cells - low glucose (1000 mg/L) not as aerobic (prefer to grow in low sugar, but will survive high)

Antibiotics commonly used: eg penicillum streptomyocin huge range which can be used AB need to be replaced- short cell life

Counting cells; cells will settle down and you can be able to see the cells to quantify them- can do this because there is a specific volume in each well (heamasotomita slide)(want to count like 100 cells) or can use an automated counter (can count total no. of dead and live cells)

cell storage so can take a break so dont have to split cells forever-- done via freezing cells (ST-weeks, LT-Years) add substance which stop ice crystals forming within the cells, puncturing holes in membrane damaging cells.. Also add more serum to buffer freezing effect may only recover 5-10% of cells

Lab 8

Reminder to self: add copyright info to Edit page within images link

Questions

1) Identify a cell line from the ATCC which was derived from the mouse

CCL-147


ATCC® Number: CCL-147™ Price: $551.00 (for-profit list price)


Designations: NB41A3

Depositors: G Augusti-Tocco

Biosafety Level:1

Shipped:frozen

Medium & Serum: See Propagation

Growth Properties: loosely adherent, multicell aggregates


2) Tissue of orgin Organism: Mus musculus (House Mouse)

Morphology: neuroblast; with elongated processes which emanate

Source:

Organ: brain Disease: neuroblastoma Cell Type: neuroblast;

Cellular Products: choline acetylase; tyrosine hydroxylase; acetylcholinesterase

Permits/Forms: In addition to the MTA mentioned above, other ATCC and/or regulatory permits may be required for the transfer of this ATCC material. Anyone purchasing ATCC material is ultimately responsible for obtaining the permits. Please click here for information regarding the specific requirements for shipment to your location.

Virus Resistance: poliovirus 1

Comments: Tested and found negative for ectromelia virus (mousepox).

Propagation: ATCC complete growth medium: The base medium for this cell line is ATCC-formulated F-12K Medium, Catalog No. 30-2004. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 2.5%; horse serum to a final concentration of 15%.

Temperature: 37.0°C

Atmosphere: air, 95%; carbon dioxide (CO2), 5%

Subculturing: Protocol: Remove medium, and rinse with 0.25% trypsin, 0.03% EDTA solution. Remove the solution and add an additional 1 to 2 ml of trypsin-EDTA solution. Allow the flask to sit at room temperature (or at 37C) until the cells detach.Add fresh culture medium, aspirate and dispense into new culture flasks.

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:4 is recommended

Medium Renewal: 2 to 3 times per week

Preservation: Freeze medium: Complete growth medium, 95%; DMSO, 5%

Storage temperature: liquid nitrogen vapor temperature

Related Products: Recommended medium (without the additional supplements or serum described under

ATCC Medium):ATCC 30-2004

recommended serum:ATCC 30-2020

recommended serum:ATCC 30-2040

References:

23251: Buonassisi V, et al. Hormone-producing cultures of adrenal and pituitary tumor origin. Proc. Natl. Acad. Sci. USA 48: 1184-1190, 1962. PubMed: 13874682 26010: Burn JH, Rand MJ. Acetylcholine in adrenergic transmission. Annu. Rev. Pharmacol. 5: 163-182, 1965. PubMed: 14290806



3) The orginal reference: <pubmed>13874682</pubmed>



Notes

Lab 8:

PC 1: physical containment level 1- lowest level undergrad allowed (waste is incinerated) PC 2, working with human material (source of infection) and work with transgenic animals (this lab has higher level of containment). there are hazards but not as high (waste is autoclaved before incinerated) PC 3: highest level containment- everything that comes in doesnt come out. dangerous biological organisms. potentially hazardous eg aids or some unknown diseases. Double entry

OGTR- (office of gene technology regulated) controled how genetically modified organisms are worked with/ in specific regulations which labs need to comply with.

Work with humans and animals needs to be ethically approved (by logding an ethics approval to Uni or hospital- need signed approval) Need ethic approval when you do primary cell work

No longer OHS, its HS as all australia complys with same regulation (need legislation 1 jan 12)

Incubator: a range of cells used for growing in different culture. flasks for growing cells and or collecting supernant. small n large. 10cm dishes each media, loosly fitting cap so gases can circulate. Well dishes ( usually put a glass cover slip for growing organisms on them) organism easily adhere to plastic

early culture was reptilian cells (easy to grow cells optimal at room temp) and chicken. dont use open benches to culture, as risk contamination.

Culturing done in sterile environments- biohood. UV light tube to radiate environment. when turned on, pump turned on and sucks out air (hepa filter of .2 micron) which is sufficent to remove most bacteria and fungi), within hood air is therefore sterile. UV light not on when working, as will kill any organism with water in them (no killing of some fungal spores) used materials are steriled (usually gamma radiation- for plastic)

overlapping lid is suffient to trap organism, some have a filter cap as well (0.2 microns)

1% bleach 80% ethanol (cleans bleach) Virasol used for virus culture preparation.. All this is chemical waste..

Biohood Class 1: doesnt protect person Biohood Class 2: not blowing air onto you- protective Biohood Class 3: never expose yourself to material, touching with the gloves

Fridges: store media, cells, etc

12:paying for things.. (some GF like $500 for a micron)

CELL LINE: is a stable cell that can reproduce in culture, will have properties each generation.

Australia doesnt have a main supplier, smaller ones (in hospital sell cord cells)

rat undifferetianted cell- need neuronal GF to make them differentiate. Tells you morphology, references of how cell line was first derived, how to grow, price, will grow as long as given right things.

Epithelial cells if form a single sheet, will stop dividing (cant be confluent)


Primary culture: derived from organism itself Used in neural research as neurons dont divides.

Adherent cells- the cells stuck to the substate (plastic dish) Suspension culture- blood cells are grown floating in the medium (often contained in siliconised and continully in motion so cells dont stick) Semi- at some stage they can stick or can be floating (monocytes -> macrophages) most difficult to grow


if cells stuck to the plate how do we get them off? use two methods: 1) mechanically remove by scraped- potential damage 2) enzymatically- using trypsin digests any protein or glycoprotein, so digests these components of the cells which are in contact with the plastic, (1min, tapping the cells) Also treat with phosphate buffer (EDTA) - removes Ca and Mg, for which alot of the adhesion molecules are required to stick.


Phase microscope- cells growing in tissue culture can be easily seen. (inverted)

Media- water inorganic salts to maintain osmolality (if hypo osmo- swelling and burst) adjusted to ph 7.4 + phenol red tells you in right ph range growing cells release metabolites and make the media acidic, media turn orange! confluent amount of cells or too long growing on same media (purple if basic) buffer system is Bicarbonate, incubators also have a specific concentration of CO2 like 5% Serum (all nutrients)- mainly fetal cow serum- need lots of factors to grow) stem cell work no serum added!! just growth factors add antibiotics- will supress any growth of bacteria (new thing, in past not added)

Incubator-- dark apart from epithelial cells most cells grow in darkness.. water for humidity (also grows fungus n moulds) 5% CO2 circulating air

Lab 7

Questions to be posted

Contribution (to date) to your group project? Project 7

Introduction: made some basic editing changes, created subtitles and formatted picture sizes, added two images:

Adrenoceptors


Adrenergic receptors within the human body

--Z3333208 19:36, 30 April 2012 (EST)

i gave group member allocated this section some basic information to add to intro as she was getting stuck. a recent comment to member " I found in one of the online book a link to the info, which you could use as a reference, but we can't use any of the images in the books... MBoC - Some G Proteins Signal By Regulating the Production of Cyclic AMP".

History: located a couple of useful, full access and easy to read articles (posted in discussion-as well as my page), i will go over to make sure have added relevant info where needed at some later stage.

I Added:

2003- Fluorescence resonance energy transfer (FRET)-based studies suggest that, at least for certain G-proteins (particularly Gi type), a conformational rearrangement might occur rather than separation of the alpha subunit and beta & gamma subunit [2]. This showed that models that may be accepted for many decades are not completely accurate depictions of the true molecular events.

2003- Analysis of the sequenced human genome (HUGO), led to the identification of over 800 GPCR genes [3]

--Z3333208 18:16, 23 April 2012 (EST)

Gene description: I added info:

The genes, ADRB1, ADRB2 and ADRB3 each code for the expression of the Beta adrenoceptors subtypes below. It has been long established that DNA mutations and genetic defects lead to disease and illness. Studies have been conducted into the rate of evolution of these genes, in order to infer whether environmental changes act as selective forces driving evolution, subsequently leading to changes in these receptors[4] . --Z3333208 09:44, 1 May 2012 (EST)

Defects with ADRB1 are the focus of this project (include a variety of cardiovascular disorders) and are discussed in Abnormal section.

--Z3333208 09:54, 1 May 2012 (EST)

Defects ADRB2 are linked with asthma (MIM 600807).

--Z3333208 09:54, 1 May 2012 (EST)

Defects in ADRB3 are associated with insulin resistance and obesity (MIM 601665).

--Z3333208 09:46, 1 May 2012 (EST)

Added a new section which i plan on working on called Receptor Agonists

My comment: Maybe we can make this a little table, and have natural endogenous agonists, and exogenous agonists, maybe even a couple of examples of antagonists (propanolol-beta blockers!) I can do this section --Z3333208 19:28, 30 April 2012 (EST)

Receptor Structure: (was my section originally- have added all info below and still working on reading some journals)

In order to have a valid and precise understanding of the mechanisms involved in activation and signal transduction pathways, it is a vital requirement that the structure of each GPCR be identified. Due to new technological advances more and more is beginning to be discovered about different structures across families and within subtypes of families.

This is a simple depiction of a standard GPCR. It shows the seven hydrophobic domains which can be seen to spam the membrane (grey)

General Classification

One key step in categorising the GPCR's came after the human genome was determined. Analysis of the human genome found that the GPCR were the largest group of membrane proteins, with approximately 800 unique types.

There are considered to be 5 families [5]:

- Rhodopsin, contains 701 members (Beta adrenergic GPCR fit into this category)

- Adhesion, 24 members

- Taste, 24 members

- Glutamate, 15 members

- Secretin, 15 members.

- There is a large portion of 'orphan' GPCR which don't fit into a specific family.

General components

MBoC - Figure 15-26 , this link provides an illustration of a simple GPCR.


  • The defining aspect of GPCR which is the basis of all receptors is that all contain seven membrane-spanning hydrophobic domains (alpha helices). Often GPCR are termed 7TM receptors or serpentine receptors due to this property.
  • The receptor is composed of a single polypeptide chain
  • Connecting the 7TM segments are 3 intracellular loops (labelled IL-1 to IL-3) and three extracellular loops (EL-1 to EL-3).
  • It is also common that there is an extracellular amino terminus (N-terminus) segment and an intracellular carboxyl terminus (C-terminus).
  • The structure of the N- and C- segments may regulated other functions besides basic agonist binding. The C-terminus usually contains serine (Ser) or threonine (Thr) residues that can be phosphorylated to increase the affinity and accelerate binding of regulatory proteins called β-arrestins [6]


This is a more detailed illustration of a standard GPCR shown simply in 2 dimension


Even though the three Beta adrenergic receptors are considered GPCR, they are structurally unique. The amino acid sequence between Beta-1, and Beta-2 Adrenoceptors differs by approximately 50 amino acid differences. These differences are even larger when compared to the alpha adrenoceptors[7].

The most variation in the GPCR comes from structural variations of: -A-terminus (greatest variation) -C-terminus -TM loop 5 -TM loop 6 The tertiary structure of this protein is a barrel shaped, this can be seen by observing the depictions of the receptors.


Another factor that causes structural variation is the range of agonist which bind to the receptor. There are a variety of ligands that bind to activate different types of GPCR's. These can be subatomic particles (photons of light-rhodopsins), ions (Ca2+), proteins and peptides, hormones, neurotransmitters. The agonist needs to interact with the active binding site on the GPCR, therefore structure will vary based on this factor. Not all GPCR express the binding site on the extracellular domain (N-terminus), this may be the case for proteins or peptide agonists, but consider Ca2+ as an agonist, such as small ion could cross the lipid bilayer and the binding site may well be within or across the TM domains. [8]


For an agonist binds, an inactive receptor state is present, this can be depicted at MBoC - Figure 15-27 . After an agonist binds this causes a conformational shape change in the receptor itself. This allows the activation of the intracellular G-proteins. This can is shown in MBoC - Figure 15-28

G-Protein Structure

In order to obtain a specific visual of certain GPCR, certain high resolution techniques are required (for example crystal screening, crystallography, fluorescence spectroscopy) . Obstacles are continually present, these include protein production, purification (without damage to GPCR), protein stability. [9]

Beta-1-Adrenoceptor Structure

Specific details about the structure of this receptor can be accessed from GPCRDB[10], information on Beta-1 .

--Z3333208 12:37, 19 April 2012 (EST)

Another database for information regarding, Beta-1 Adrenergic Receptors from Drug Bank[11].

--Z3333208 18:28, 30 April 2012 (EST)

Provided team mate with an external link in their section of normal pathway.. This link also has a Movie.. MCB - Figure 20-16- Activation of adenylyl cyclase following binding of an appropriate hormone (e.g., epinephrine, glucagon) to a Gs protein – coupled receptor a link to an illustration of the activation of adenylate cyclase. A movie of extracellular signalling is also present.

Regulatory mechanism: I originally added this as a section, however another team member has taken it on board and will integrate with her original section..

Provided a couple of journal articles to member completing abnormal function section.

Added a new section, called Beta Blockers. (will make suggestion to combine with receptor agonists producing a table)

Added a few glossary terms (approx 6)

Set up reference section

working on table,, which is pretty hard to do in wiki!

Subtype Natural Agonist Synthetic Agonist Non-specific Beta-blocker Specific Beta-blocker
Beta 1

A, NA

ISO

Propranolol

.

Beta 2

A, NA

ISO

Propranolol

..

Beta 3

A, NA

ISO

Propranolol

...


--Z3333208 22:32, 2 May 2012 (EST)

Ongoing work on assignment

MIM B2

MIM B1


<pubmed>22378779</pubmed>

Info on regulation section



<pubmed>21772288</pubmed> (full text)

<pubmed>17952055</pubmed> (abstract only)

info on receptor structure specifically Beta 2.


<pubmed>21915263</pubmed>

(full text-images can be used from PLoS)

AR B1 and ARB2 shown with agonists


The genes, ADRB1, ADRB2 and ADRB3 each code for the expression of the Beta adrenoceptors subtypes below. It has been long established that DNA mutations and genetic defects lead to disease and illness. Studies have been conducted into the rate of evolution of these genes, in order to infer whether environmental changes act as selective forces driving evolution, subsequently leading to changes in these receptors[12] .

Lab 6

Graph

Differences in Phenotypes in Control & TM4 cells.jpeg

Homework

1)Do you see a difference in phenotype (morphology) between Tm4 overexpressing and control cells?

The control group has more cells in the broken fan phenotype, with less cells developing into the later phenotypic stages. The pronged and stringed phenotypes show very low level for the control cells (greater than 10% less when compared to TM4). The TM4 cells are expressed in a much lower proportion in the broken fan phenotype (40% less-compared to control). The TM4 cells then increase in the later developing phenotypes. The have greater amount of cells than the control in stumped, pronged, stringed phenotypes. There was no cells in the fan phenotype for both, but these are rare to find. The pygnotic group showed similar amounts in both TM4 and control (about 4% more in control).

2)If so, how could Tm4 overexpression lead to this difference?

As TM4 has cells in greater number in the later developing phenotypes. TM4 may be actively involved in the events of neuritis growth in these later phenotypic stages. It may be the TM4 may inhibit the broken fan phenotype as there is far fewer cells expressing this morphology.

Differences between Genotype A and B

TM4:

  • More branching, more processes
  • Longer, thicker, more florescent processes
  • Appear in groups more rarely than control

Control:

  • Less branching and processes
  • Thinner processes, shorter
  • Cells more often clump together
  • Cells appear to be under going mitosis a lot more
  • A lot of cells in earlier development phenotypes


cAMP induced cells

3)Do you see a difference in phenotype (morphology) between Tm4 overexpressing and control cells?

Differences between Genotype A and B

TM4:

  • More branching processes
  • Individual processes are shorter and wider
  • Cells appear in groups more than control
  • Soma are more bulgier and rounder

Control:

  • Less branching processes
  • Individual processes are long and thin, extend in a longitudinal fashion
  • Cells appear to be less likely clumped together
  • Soma are often more flattened and smaller

4)If so, how could Tm4 overexpression lead to this difference?

TM4 appears to be involved in the neroblastoma morphological cell development. With more cells found in the pronged and stringed phenotypes than the control. And the TM4 over expressing cells cause processes to branch more often during the growth of the neuritis.

TM-4 may be involved in the motile events of neurite growth and synaptic plasticity. [13]


[14]

Work on project

<pubmed>20028316</pubmed>

  • An article about history of GPCR generally

<pubmed>16902930</pubmed>

<pubmed>16402120</pubmed>

  • Another 2 appear easier to read


READ

<pubmed>1974837</pubmed>


Own Lab Notes

Actin cytoskeleton

Neuroplastoma cell line. Analyse processes in neurons. Neuroblastoma be cultured. removal of serum, and cAMP addition to culture to induce process formation look at early stages of neural cell producing processes. somatodendritic compartment: soma and dendrites introduce protein into cell, observe effect of neuronal function. Tropomysion, actin associated protein. A range of TM, generated by 4 different genes TG1-TG4. Transfection, using plasmids. Green actin, red Tm

Lab 5

Work on group project

<pubmed>17188232</pubmed>

article contains Obstacles for obtaining GPCR Structures and COMMON STRUCTURAL FEATURS OF GPCRS Read and used!.


<pubmed>16595179</pubmed>

article for signalling, receptor silencing (via arrestin) generalised article.. I think we should discuss difficult pathways as simple general concepts..


G-protein-coupled receptor structure: what can we learn? A review which simply goes through importance of finding out the structure, but mainly discusses beta 2.



In order to have a valid and precise understanding of the mechanisms involved in activation and signal transduction pathways, it is a vital requirement that the structure of each GPCR be identified. Due to new technological advances more and more is beginning to be discovered about different structures across families and within subtypes of families.


One key step in categorising the GPCR's came after the human genome was determined. Analysis of the human genome found that the GPCR were the largest group of membrane proteins, with approximately 800 unique types. There are considered to be 5 families: - Rhodopsin, contains 701 members - Adhesion, 24 members -Taste, 24 members - Glutamate, 15 members - Secretin, 15 members. - There is a large portion of 'orphan' GPCR which don't fit into a specific family.

above reference PMID: 12761335


The defining aspect of GPCR which is the basis of all receptors is that all contain seven membrane-spamming hydrophobic domains. Often GPCR are termed 7TM receptors due to this property. It is also common that the amino terminus segment is extracellular and the carboxyl terminus is intracellular. The most variation in the GPCR comes from structural variations of: -A-terminus (greatest variation) -C-terminus -TM loop 5 -TM loop 6

Another factor that causes structural variation is the range of agonist which bind to the receptor. There are a variety of ligands that bind to activate different types of GPCR's. These can be subatomic particles (photons of light-rhodopsins), ions (Ca2+), proteins and peptides, hormones, neurotransmitters. the agonist needs to interact with the active binding site on the GPCR, therefore structure will vary based on this factor. Not all GPCR express the binding site on the extracellular domain (N-terminus), this may be the case for proteins or peptide agonists, but consider Ca2+ as an agonist, such as small ion could cross the lipid bilayer and the binding site may well be within or across the TM domains.

In order to obtain a specific visual of certain GPCR, certain high resolution techniques are required (for example crystal screening, crystallography, fluorescence spectroscopy) . Obstacles are continually present, these include protein production, purification (without damage to GPCR), protein stability.

--Z3333208 23:00, 18 April 2012 (EST)


READ http://mend.endojournals.org/content/24/1/261.long PMID: 20019124 contains image of b2-- can't use don't have permission from journal.. but easy to read article!

Lab 4 Immunochemistry

Lab 4

Own Notes:

Lab 4 two heavy chains joined by disulfide bridges. two light chains. interaction between heavy and light chains, creates a variable region. area which structure /antigen binds. two binding sites- can bind two epitopes or proteins. lower region with two heavy chains doesnt bind epitope. can bind a Fc protein. Fab fragment which still has antibody binding domain in it. Research antibodies are in the Igg or Ig 1 family. B cells secrete antibody- each b cell will only ever make one type of Ig, but make thousands of clones /copies all who have been vaccinated have been experimented on. to develop antibodies to the antigen (proteins which introduced into the body) B memory cells are primed and ready to produce antibodies to attack that specific antigen up on second exposure to antigen. 2 or 3 injection, serum will have high levels of antibodies specific to that antigen. But if animal had been previously exposed to an antigen, then antibodies for such an antigen will be present. Antibodies would be present in centrifuged plasma component. Antigen has to be non-toxic, and needs to be injected into muscle tissue to store in body for longer (depot in muscle). bacterial adjevit- stimulates immune system

Different b cells, each will recognize at various sites, epitopes of the antigen, and subsequently each will produce a single antibody (binding at different location) but each will produce a different antibody. -polyclonal antibody generation recognises a range of epitopes- so adding a similiar antigen may stimulate another antibody by binding to a similiar epitope. this will cause a % of the antibodies attaching to another epitope (non-specific) is called a background. Poly- good amplify signal but non-specific binding may occur, limited to the amount of times you can bleed the animal to test for serum (limited by amount of serum which can be produced) Western blotted membrane- less bands means antibody is more specific.. more bands- less specific.

Producing a single antibody clone: monoclonal Primary antibody, binds to the protein of interest. Expensive. Dont have to label it. Secondary antibody (Igg) binds to the primary antibody (from another species) Cheap. Can florscently/enzymatically label it- only label secondary.


Activities

Musashi Protein

It is a real protein, it actually is a family of RNA-binding proteins. It is expressed in the nervous system. The first protein from this family was discovered in Drosophila. It plays a role in regulating the cell division of ectodermal precursor cells through the translational regulation of target mRNA. It is a neural RNA-binding protein that is strongly expressed in fetal and adult neural stem cells (NSCs). It contributes to the self-renewal of NSCs. Musashi-2 has been postulated to act in the same manner as Musashi-1 in regulating NSCs. Whether Musashi-1 and -2 proteins have target mRNA(s) other than m-Numb mRNA remains an open question.

Primary Article which provided the above information:

Hideyuki Okano, Hironori Kawahara, Masako Toriya, Keio Nakao, Shinsuke Shibata, Takao Imai,Function of RNA-binding protein Musashi-1 in stem cells. A review article. Experimental Cell Research Volume 306, Issue 2, 2005, Pages 349–356 Article can be found at http://www.sciencedirect.com/science/article/pii/S001448270500090X


  • First discovered 1997 in mice [15].
  • The human musashi protein is 362 amino acids. This information was found at the website below:

http://www.ncbi.nlm.nih.gov/protein/BAA33962.1

Primary Antibody

Name: Anti-Musashi 1 / Msi1 antibody (ab21628)

Rabbit polyclonal to Musashi 1 / Msi1

Reacts with Mouse, Human

Predicted to work with Rat, Xenopus laevis

Immunogen: Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Musashi 1 / Msi1.

Clonality: Polyclonal

Isotype: IgG

Tissue specificity: Detected in fetal kidney, brain, liver and lung, and in adult brain and pancreas. Detected in hepatoma cell lines

Cellular localization: Cytoplasm. Nucleus.

Test Applications: -Immunocytochemistry/ Immunofluorescence -Western blot

Concentration: Batch dependent within range *100 µg at 1-1.4mg/ml

Information found at http://www.abcam.com/Musashi-1-Msi1-antibody-ab21628.html


General example; Dilute 1/50 a low concentration of antibody in original sample. Using above range will get non-specific binding.


Secondary Antibody

Goat anti-Rabbit IgG

Antigen: IgG

Clonality: Polyclonal

Host: Goat

Reactivity: Rabbit

Conjugate(stuck to): Alexa Fluor 488

Concentration: 2mg/1ml

Applications: Western Blotting (WB), ELISA, Immunoprecipitation (IP), Immunofluorescence (IF), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunohistochemistry (Formalin-fixed Sections) (IHC (f)), Flow Cytometry (FACS)

Information found at http://www.antibodies-online.com/antibody/727475/Goat+anti-Rabbit+IgG+Alexa+Fluor+488/

Lab 3 Preparation/Fixation

Lab 3 --Z3333208 15:07, 22 March 2012 (EST)


Own notes

Biological material infected scapel then cut and formed wound. Firstly wash with water, find someone with first aid. Then you need to make a report of the incident. UNSW if deemed hazardous will require a blood test.(will test for the material in your body- so determine levels in your body- this is only relavent if chemical or biological material is testable)

Techniques for tissue and cell fixation.

1. Fresh Frozen 2. Precipitation 3. Aldehyde Cross-linked

1. Biological material not be fixed, will freeze (in a way to prevent ice crystal damage- which destroys cell integrity)and slice and observed. Materials biological and enzymatic material wills still remain active for a certain period of time. Retain a certain amount of activity, reserve epitopes (site on surface of protein which recognises the antibody and the antibody binds to it. Lipids normally removed. Cryotomes are minus 20 degrees, tissue remains frozen. PPE required for working with material.. Like salami sliced in deli.. Slice kept a room temp, thin section on bio material is frozen, and stuck onto the slide.

2. Preserves the tissue by precipitating proteins with an organic solvent. Draws water out of cell, ie shrinking cells causes artefacts. FIXATION ALTERS SIZE OF MATERIAL (often shrinkage). 3. Prevents degrading of proteins, will reserve structure (collagen). formalin (intermediate strength) can be made to be isoosmotic to the cell to prevent rupture. Para formaldehyde -weakest of the three (powder repiratory hazard), doesnt cross link as strong, best for preserving epitomes of cells. Use fresh. longer left in solution, cross linking will increase and then antibodies will not be accessable for immunological staining. Gluteraldehyde STRONGEST cross linker, used for EM work.

Detergents, remove lipid from fixed material- can permeate the cells.

Activities

Locate a current SDS for one of the fixatives described in today's lab. Identify the properties and hazards associated with that chemical.

Data obtained on Chemalert (data from 31 January 2008) [[1]]

Fixative- Formalin

Properties

Appearance: CLEAR COLOURLESS LIQUID

Solubility (water): SOLUBLE

Odour: PUNGENT IRRITATING ODOUR

Specific gravity: 1.1 (Approximately)

pH: 2.4 to 4.0

% Volatiles: > 44 %

Flammability: CLASS C1 COMBUSTIBLE

Vapour density: 1.04 (Air = 1)

Flash Point: 85°C (Approximately)

Boiling point < 100°C

Upper Explosion Limit 73 %

Lower Explosion Limit 7 %

Autoignition temperature 430°C


Hazards Toxic - corrosive. This product has the potential to cause adverse health effects. Use safe work practices to avoid eye or skin contact and inhalation. Contact may result in burns with possible tissue damage. May cause sensitisation by skin contact. Formaldehyde is classified as a confirmed human carcinogen (IARC Group 1). Chronic exposure may result in cell mutations, reproductive system effects, liver damage and insomnia. Chronic exposure to methanol may result in optic nerve damage.


Eye Corrosive - irritant. Contact may result in irritation, lacrimation, pain, redness, corneal burns and possible permanent damage.


Inhalation Toxic - corrosive. Over exposure may result in mucous membrane irritation of the respiratory tract, coughing, chest pain and sensitisation with asthma-like symptoms, breathing difficulties, pulmonary oedema and convulsions at high levels. Chronic exposure may result in liver damage and fertility effects (sperm count and viability, increase in spontaneous abortions).


Skin Corrosive. Contact may result in irritation, redness, pain, rash, dermatitis and possible burns. May cause sensitisation by skin contact.


Ingestion Toxic - corrosive. Ingestion may result in gastrointestinal ulceration, nausea, vomiting, abdominal pain, acidosis and diarrhoea (bloody). Ingestion of large quantities may result in liver and kidney damage, pulmonary oedema, unconsciousness and death which may be delayed.


Identify 4 papers required for your group work project. Cite on the Group Project discussion page and also on your own Individual page. Add one sentence for each as too why they are relevant to your group topic

1 <pubmed>20351116</pubmed> A primary article linking defective GPCR to heart failure. This can be used for abnormal receptor function section


2 <pubmed>12648290</pubmed> A review article about how Beta Gpcr linked to asthma, can also be used for the abnormal receptor functioning section.


3 <pubmed>16460808</pubmed> This is also a review. It initially gives a brief and then detailed information on arrestins which are proteins which bind to GPCR and inhibit their action. They are vital in signal transduction pathways, and I think that it will be necessary for us to discuss their role.


4 <pubmed>9398661</pubmed> The article above is a primary article which investigated the location of GPCR in the plasma membrane. I think it will be good to add in the intro sections, that overall there was no specific location for the receptors morphologically.


Also found this article with few good images about the actual configuration of the receptor in different states. We can use such images along side the section for structure. <pubmed>22031696</pubmed>


And the following articles i think may be useful, but are more complicated i thought id post them up maybe a few of us can have a go at understanding them better. They are generally concerned with beta arrestins, internalisation, regulation of the signal transduction pathway.

<pubmed>15634674</pubmed> <pubmed>9145918</pubmed>

Lab 2 Microscopy Methods

Lab 2 --Z3333208 15:13, 15 March 2012 (EST)

Activities

In class task: Upload an image

Satelite cell.jpg

Identify a reference article that uses the "superresolution" microscopy technique.

<pubmed>22006021</pubmed> PMID 22006021

What did the paper show that normal microscopy could not show.

Mitochondria store their own DNA in structures called nucleoids, such structures have previously not been able to be visualised well enough to study. Superresolution microscopy has allowed these nucleoids to be clearly imaged. It has been shown that nucleoids differ in shape (roughly ellipsoid) and size, but are distinctly located near the inner mitochondrial membrane (IMM) whereby the cristae of the IMM wraps part of the membrane around them. By being able to look deeper into the nucleoid, it has also been noted that the mitochondrial DNA is highly condensed within these structures. These new findings are allowing scientists to better understand the mechanisms and relationships between mitochondrial DNA and the nucleoid.

Nucleoids in association with mitochondrial membrane.jpeg

Lab 1 Introduction

Lab 1 --Z3333208 15:30, 8 March 2012 (EST)

Activities

External link with a name coming up as the link, put website in [] and after the name of website write the name GOOGLE

External link, no special name, just type website. http://www.google.com.au

Internal link; using double bracket [[, name of link in the website, vertical line |, name you want to appear followed by double bracket Lecture 2 Lecture 3

[16]

Lab Attendance

--Z3333208 15:30, 8 March 2012 (EST) Lab 1

--Z3333208 15:13, 15 March 2012 (EST) Lab 2

--Z3333208 15:07, 22 March 2012 (EST) Lab 3

--Z3333208 14:06, 29 March 2012 (EST) Lab 4

--Z3333208 15:10, 5 April 2012 (EST) Lab 5

--Z3333208 14:08, 19 April 2012 (EST) Lab 6

--Z3333208 14:30, 26 April 2012 (EST) Lab 7

--Z3333208 14:12, 3 May 2012 (EST) Lab 8

--Z3333208 15:36, 10 May 2012 (EST) Lab 9

--Z3333208 14:14, 17 May 2012 (EST) Lab 10

--Z3333208 14:41, 24 May 2012 (EST) Lab 11

--Z3333208 14:04, 31 May 2012 (EST) Lab 12

References

  1. <pubmed>22629434</pubmed>
  2. <pubmed>14673086</pubmed>
  3. <pubmed>12761335</pubmed>
  4. <pubmed>19576569</pubmed>
  5. <pubmed>12761335</pubmed>
  6. <pubmed>2163110</pubmed>
  7. Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. G Protein –Coupled Receptors and Their Effectors.
  8. <pubmed>17188232</pubmed>
  9. <pubmed>17188232</pubmed>
  10. GPCRDB: information system for G protein-coupled receptors. Nucleic Acids Res. 2010 Nov 2 Vroling B, Sanders M, Baakman C, Borrmann A, Verhoeven S, Klomp J, Oliveira L, de Vlieg J, Vriend G
  11. DrugBank: a knowledgebase for drugs, drug actions and drug targets. Wishart DS, Knox C, Guo AC, Cheng D, Shrivastava S, Tzur D, Gautam B, Hassanali M. Nucleic Acids Res. 2008 Jan;36(Database issue):D901-6. PMID: 18048412
  12. <pubmed>19576569</pubmed>
  13. <pubmed>7876361</pubmed>
  14. <pubmed>11571370</pubmed>
  15. <pubmed>9047340</pubmed>
  16. <pubmed>19564476</pubmed>