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Nuclear folds.jpg Nuclear Folds http://www.ncbi.nlm.nih.gov/pubmed/5801425

External Links

JOVE

Internal Links

Cell Nucleus

Lecture 4

Lab 2 - Microscopy

As presented by Dani et al. (2010), the introduction of superresolution microscopy has enabled significant improvements in brain tissue studies. Previously, studies of brain tissue has had access to only images from electron microscopy and therefore the location of proteins within dense brain tissue was not possible. Superresolution microscopy has enabled high quality imaging the localization of various proteins embedded in brain tissue synapses.

Reference Adish Dani, Bo Huang, Joseph Bergan, Catherine Dulac, Xiaowei Zhuang Superresolution imaging of chemical synapses in the brain. Neuron: 2010, 68(5);843-56 PubMed 21144999


--Mark Hill 12:43, 20 March 2012 (EST) This is a reasonable explanation, you could have mentioned the specific proteins in this study. Also the image below should have a legend and the reference linked. This will be discussed in Lab 3.

Lab 3- Fixation

Paraformaldehyde

Properties

Appearance Form powder

Odour pungent

Safety data pH 4.0 - 5.5

Melting point 120 - 170 °C - lit.

Boiling point no data available

Flash point 70 °C - closed cup

Flammability (solid, gas) The substance or mixture is a flammable solid with the subcategory 2.

Ignition temperature 370 °C

Lower explosion limit no data available

Upper explosion limit no data available

Density 0.88 g/cm3 at 25 °C

Water solubility insoluble

Hazards

Low to moderate toxicity - irritant. This product has the potential to cause adverse health effects with over exposure. May release formaldehyde, which is a skin and respiratory sensitiser and classified as a confirmed human carcinogen (IARC Group 1).

Eye Severe irritant. Contact may result in irritation, lacrimation, pain, redness and blurring or dimness of vision. Prolonged contact may result in corneal burns and possible permanent damage.

Inhalation Irritant. Over exposure to dust may result in mucous membrane irritation of the respiratory tract, coughing and breathing difficulties. Upon decomposition, this product may generate formaldehyde, which is a potential sensitising agent and may result in asthma-like symptoms.

Skin Irritant. Contact may result in irritation, redness, rash and dermatitis. Prolonged or repeated contact may result in burns. May be absorbed through skin with harmful effects. May cause sensitisation by skin contact.

Ingestion Low to moderate toxicity. Ingestion may result in gastrointestinal irritation, nausea, vomiting, headache and diarrhoea. However, due to product form ingestion is considered unlikely. Maintain good personal hygiene standards.

reference: SIGMA-ALDRICH MSDS information: Version 4.5 Revision Date 13.12.2011 Print Date 29.03.2012 Chemalert


Articles for Group Project - Group 1

1 Marina Grigorova, Margus Punab, Birutė Zilaitienė, Juris Erenpreiss, Kristo Ausmees, Valentinas Matuleviĉius, Igor Tsarev, Niels Jørgensen, Maris Laan Genetically determined dosage of follicle-stimulating hormone (FSH) affects male reproductive parameters. J. Clin. Endocrinol. Metab.: 2011, 96(9);E1534-41 PubMed 21733993


The effects of the low level of follicle stimulating hormone (FSH) on the male reproductive system. It shows the indirect role of testosterone and FSH.


2 M Scobey, S Bertera, J Somers, S Watkins, A Zeleznik, W Walker Delivery of a cyclic adenosine 3',5'-monophosphate response element-binding protein (creb) mutant to seminiferous tubules results in impaired spermatogenesis. Endocrinology: 2001, 142(2);948-54 PubMed 11159868


Examination of the role of cAMP response element-binding protein (CREB)in spermatogenesis.


3 W P Benten, M Lieberherr, G Giese, C Wrehlke, O Stamm, C E Sekeris, H Mossmann, F Wunderlich Functional testosterone receptors in plasma membranes of T cells. FASEB J.: 1999, 13(1);123-33 PubMed 9872937


The presence of testosterone receptors on T-cell membranes suggesting the potential of testosterone being involved in immunity.


4 Robert W Holdcraft, Robert E Braun Hormonal regulation of spermatogenesis. Int. J. Androl.: 2004, 27(6);335-42 PubMed 15595952

Review

The role of testosterone in spermatogenesis.

Lab 4

Nicole A Siddall, Eileen A McLaughlin, Neisha L Marriner, Gary R Hime The RNA-binding protein Musashi is required intrinsically to maintain stem cell identity. Proc. Natl. Acad. Sci. U.S.A.: 2006, 103(22);8402-7 PubMed 16717192

Y Kaneko, S Sakakibara, T Imai, A Suzuki, Y Nakamura, K Sawamoto, Y Ogawa, Y Toyama, T Miyata, H Okano Musashi1: an evolutionally conserved marker for CNS progenitor cells including neural stem cells. Dev. Neurosci.: 2000, 22(1-2);139-53 PubMed 10657706

Uniprot

Musashi are real proteins, they are a group of proteins that are RNA binding. They are present in proliferative cells such as Drosophila testis. Function: involved in germ stem cell development in the central nervous system. Sequence length varies between 328-362 AA depending on where it is found for example, in rats, humans or mice.


Musashi 2 antibody

primary antibody

rabbit raised polyclonal antibody

application: detects MSI2 by western blotting at 1 - 2 μg/ml.

dilution: 1 - 2 μg/ml

specificity: reactive in human, mouse and rat.

data sheet for MSI supplier LIFE RESEARCH

Secondary antibody -anti rabbit IgG antibody [1]

Lab 6

Biojbi.JPG

1. Do you see a difference in phenotype (morphology) between Tm4 over-expressed and control cells?

Genotype A- Tm4

- more processes

- more branching

- longer processes

- thicker processes

Genotype B- Control

- less processes

- less branching

- shorter processes

- thinner processes

2. If so, how could Tm4 over expression lead to this difference?

The higher number of pronged and stringed phenotypes in the Tm4 samples compared to the control suggests that Tm4 causes an increased expression of these phenotype. Tm4 may have a an inhibitory effect in regards to the pygnotic phenotype as Tm4 over-expressed cells were not present compared to the control cells.


Differences between Genotype A and B (induced with cAMP)

Genotype A

- more processes

- shorter processes

- brighter coloured structures

- rounder nuclei

- structures are more closely grouped

Genotype B

- less processes

- longer processes

- dense nuclei that can be bright blue coloured

- less grouping of structures


2. If so, how could Tm4 over expression lead to this difference?



As discussed in the review and article below, the involvement of tropomyosin and its various isoforms is seen in the morphology of neurons. Specifically it's involvement in neuron growth and the various isoforms found in certain areas of a neuron such that enable this.

C Dufour, R P Weinberger, P Gunning Tropomyosin isoform diversity and neuronal morphogenesis. Immunol. Cell Biol.: 1998, 76(5);424-9 PubMed 9797462


R Weinberger, G Schevzov, P Jeffrey, K Gordon, M Hill, P Gunning The molecular composition of neuronal microfilaments is spatially and temporally regulated. J. Neurosci.: 1996, 16(1);238-52 PubMed 8613790


Lab 7

contributions to the group project:

The binding of testosterone to an androgen receptor within a cell

Testosterone is an androgen hormone that plays an important role but is not limited to, male reproductive development, differentiation and spermatogenesis. Testosterone has been shown to act in two main pathways often referred to as the classical and non-classical pathway. Through the classical pathway, testosterone acts via the androgen receptor (AR). These receptors can be found in high numbers in the testis on sertoli cells and leydig cells in seminiferous tubules. [12] [edit] The Classical Pathway

Through this pathway, androgen hormones such as testosterone or dihydrotestosterone (DHT) bind with an AR resulting in a conformational change in the AR structure. DHT is a converted form of testosterone that is made by 5a-reductase, an enzyme found in the cytoplasm. Upon binding, of either hormone to the AR, an activated complex results via the involvement of heat shock proteins(2). The transformed androgen-receptor complex enables greater binding affinity of the structure to DNA hormone protein elements found in the nucleus, in which are necessary for gene transcription. [13]

The non- classical pathway

According to recent research, the androgen hormones including testosterone has been suggested to act via different signaling pathways. These include the mitogen-activated protein (MAP) kinase and its affect on cAMP response element binding protein (CREB). [14] [edit] Structure of the Androgen Receptor

The androgen receptor is comprised of various domains that are similar to other receptors of the same steroid family classification.

Features of the androgen receptor include: [15]

- Approximately 919 amino acids in length (3)

- N-terminal domain

- DNA- binding domain (DBD)

- Hinge region that links the DBD and LBD regions together

- Ligand or androgen binding domain (LBD)


--Z3332676 23:07, 2 May 2012 (EST)

Lab 8

1. Identify a mammalian cell line in the ATCC catalogue (and add a link)

Name of cell line: CHO-K1 ATCC Catalog

ATCC® Number: CCL-61™


Designations: CHO-K1

Depositors: TT Puck

Biosafety Level: 1

Shipped: frozen

Medium & Serum: See Propagation

Growth Properties: adherent

Organism: Cricetulus griseus (chinese Hamster)

Morphology: epithelial-like


Source: Organ: ovary

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.

Isolation: Isolation date: 1957

Applications: transfection host

Virus Resistance: poliovirus 2; modoc virus; Button Willow virus

Cytogenetic Analysis: Chromosome Frequency Distribution 50 Cells: 2n = 22. Stemline number is hypodiploid.

Gender: female

Comments: The CHO-K1 cell line was derived as a subclone from the parental CHO cell line initiated from a biopsy of an ovary of an adult Chinese hamster by T. T. Puck in 1957.

The cells require proline in the medium for growth.

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 10%.

Temperature: 37.0°C

Subculturing: Protocol:

  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
  3. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
     Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
  4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
  6. Incubate cultures at 37°C.


Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:8 is recommended

Medium Renewal: Once or twice between subculture

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

Storage temperature: liquid nitrogen vapor phase

Related Products: Recommended medium (without the additional supplements or serum described under ATCC Medium):ATCC 30-2004

recommended serum:ATCC 30-2020

References: 22224: Puck TT, et al. Genetics of somatic mammalian cells III. Long-term cultivation of euploid cells from human and animal subjects. J. Exp. Med. 108: 945-956, 1958. PubMed: 13598821 (full references are shown through external link)

2. Identify the original tissue of origin of that cell line.

Ovary

3. Identify the original paper that characterised the properties of that cell line.

Original paper: T T PUCK, S J CIECIURA, A ROBINSON Genetics of somatic mammalian cells. III. Long-term cultivation of euploid cells from human and animal subjects. J. Exp. Med.: 1958, 108(6);945-56 PubMed 13598821


Lab 9

Group Reviews

Group 2

Pros

    • good clear tables
    • pictures in abnormal function are great for giving the reader a visual idea of the disease.
    • internal link to glossary was good
    • good balance of pictures and text

cons

    • structure of receptor info?
    • incomplete glossary
    • pathway needs to be more in depth.
    • structure of some sentences were a little hard to understand on first reading i.e Since VEGFR-1 is the only receptor, which does not lead to mitogenesis, VEGF binding to VEGFR-1 competes with mitogenic action that VEGF bound to VEGFR-2 and 3 would result to.

Possible improvements:

    • For someone who has little/no previous knowledge of the topic it was a little hard to understand. This could be fixed by clearer organization of writing and easily fixed by a complete glossary. (Which I understand was probably just forgotten)

Group 3

Pros

  • introduction was very informative, interesting, pointed out key points
  • good history section
  • language was simplified and clear

Cons

  • not engaging/interesting
  • not enough pictures
  • layout makes reading hard (all writing not much else)
  • no subheadings
  • no references present
  • major headings should include when ‘abnormality occurs’ if possible.

Possible improvements:

  • dot points about information on the FasL protein to make it easier to read
  • history put into a table to make the page more interesting
  • clarify which proteins are most important out of the list of proteins involved.
  • reference list and glossary needs to be updated. Correct in-text citations needed.
  • subheadings to break up writing and add style and organisation to layout
  • pathway should be more in depth i.e how extrinsic apoptosis works, maybe as a step-process summary for easy reading.


Group 4

Pros

  • good table and pictures
  • proteins involved was clear and easy to read.
  • there was a balance of writing and pictures in protein and receptor & normal func. section
  • normal function section was good, clear, good layout & info.

Cons

  • some sections were lacking more in depth info/pics when compared to other sections
  • an overall short webpage/assignment.

Possible improvements:

  • what is the involvement/function of modifier proteins? More depth in their involvement ,structure of the protein if possible would be good.
  • abnormal function heading- to show a comparison of when it works and when it doesn’t.
  • a complete glossary
  • a consistent overall layout to make the whole page appealing to readers rather than some sections over others.
  • history table seems incomplete- did anything happen between 1930s-1978? 40 years unaccounted for.


Group 5

Pros

  • great extensive history section
  • good layout that was very easy to read
  • most sections very well cited/referenced
  • great overall,polished project with interesting info

Cons

  • diseases associated section not cited properly

Possible improvements

  • basic info about b-catenin i.e. structure. The picture is great at showing the structure but having this info reiterated in writing would make it clearer.
  • embryonic development headings seems a little out of place. Should it be under a current research section? With other current research taking place/recent findings (not a big deal)

Group 6

Pros

  • headings makes it clear, easy to read
  • overall well written project
  • pictures are relevant to the project
  • correctly referenced

Cons, needs improving

  • not enough pictures
  • pictures don’t have a legend attached to them
  • glossary should be more extensive
  • not sure if headings are required in the introduction
  • read through project to make sure there are no spelling, grammatical errors
  • more pictures to make it more interesting
  • remove ‘introduction’ subheadings in proceeding sections because its unnecessary

Group 7 Pros

  • direct internal links to pictures are good, makes it easy to locate related pictures
  • good tables
  • properly cited
  • well researched looks like you guys put a lot of effort into it.

Possible improvements

  • I think the picture descriptions should be moved to the picture’s page rather than referred directly on the main project page. The main page can still have a general overall summary of the image as it does seem relevant, maybe just remove the direct references to the image. The ‘adrenoreceptor’ image should probably have a legend, brief description also. specifically steps in the intro for relating to the GPCR process-student image
  • receptor structure section well researched and extensive, good relevant pics
  • history section could be improved by putting it into a table to make it more appealing to readers.
  • maybe incorporating dot point format as well as the paragraphs you already have for signaling pathway section. This will make it easier to read.
  • check spelling and grammatical errors.
  • bolded glossary words

Group 8 pros

  • good layout, subheadings make it easy to read
  • writing flows

Possible improvements

  • more pictures
  • normal function section doesn’t have references
  • maybe add do your history section in a table format (when it gets done)
  • spell check and grammar check
  • more extensive glossary


Group 9

Pros

  • good extensive history section

Possible improvements

  • general structure of the gene, receptor, basic info about it in dot points could help
  • do pathways have different names? Maybe organizing work under the different pathway names (in pathway section) rather than referring to first, second, third pathways
  • a legend for important abbreviations in pictures would help better understanding on the topic.


--Mark Hill 12:35, 17 May 2012 (EST) I like the pros/cons formatting and you have also included good specific rather than general comments.


Lab 12 - Microarray

1.Identify a current technique used in gene sequencing.

next generation sequencing

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

Christopher Kobierzycki, Bartosz Pula, Andrzej Wojnar, Marzena Podhorska-Okolow, Piotr Dziegiel Tissue microarray technique in evaluation of proliferative activity in invasive ductal breast cancer. Anticancer Res.: 2012, 32(3);773-7 PubMed 22399591

[2]

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

In the study above,the comparison of the tissue microarray samples and whole specimen samples were carried out on breast tissue infected with breast cancer. Tissue microarray blocks were used to examine ductal breast cancer growth. By using tissue microarrays, specific markers involved in proliferation of the cancer were looked at. These included the Ki-67 antigen and the MCM-2 protein. It is believed that these two markers have a relationship with each other and are both expressed in tissues infected with breast cancer.