Ann Bohannon-Stewart
Research Class Report
Professor: Dr. Johnson
Report on Dr. Whalen’s research

Tues. Nov 3rd 2009
I went to see Dr. Whalen to see when I could start research for her…She showed me around the lab, and some of the equipment used in her lab…and we agreed Tues at 800am would be a good time. This will be an all day lab time 5pm.

Tues Nov. 10th 2009
I went to Dr. Whalen’s lab and started working on something called the Rosettsep Procedure. Which takes human NK Cell Enrichment cocktail of whole blood is ordered from another lab and centrifuges it, 2nd we took out the white buffy coat of white blood cells with a pipette. Next we added PBS phosphate buffer and centrifuged for 10 mins. Take out and remove extra PBS layer on top and add cell culture media to the cells again….than add dye to 10ul of cell solution, and place on slide grid…Under the microscope count the number of cells in 1 grid and multiply the number by 10^4.
results=3.78x10^4

Ann Bohannon-Stewart
Class: Biochemistry
Professor: Dr. Whalen
Summery report:

Enhancing the Activity of a Protein by Stereospecific Unfolding


     Insulin is a protein hormone structure that can be changed by making a substitution of (Ala) where (PheB24) is in the side chain. In this article they claim that the substitution of protein D-Ala of insulin is destabilizing to insulin, but the (Ala) protein diastereomer enhanced some of the activity with segmental unfolding of the strand. The article goes on to explain photoactivable derivatives that have L- or D-para-azido-Phe are able to link to the insulin receptor with better D-specificefficiency, and give exposure of hydrophobic bonding in the analogs and make them able to mix with accelerated fibrillation, a form of aggregation-coupled misfolding that is associated with cellular toxicity. When you trade out PheB24 for Ala, you see a changed effect of both the D and L substitution. This article explains both effects of the Stereospecific unfolding of the protein.
     The article explains how insulin works and that insulin is a globular protein hormone made up of 2 chains. The two chains are, A chain which has 21 residues and B chain which has 30 residues. Now inside of the B chain is the area where many conformational changes take place, and the area of interest or (PheB24) is in the B side chain.

      If you look on page 2 of the article it will show you a picture of insulin in it’s 2 different chains which is label A and B. In side chain B it shows Phe B24 as an arm that holds in a folding shape pocket of the protein insulin, and it tries to show how L-Ala and D-Ala will position when Phe B24 is removed and replaced with Ala L and D. The pictures show that its storage form, requires engagement of a detachable arm at an extended receptor interface. Because this active conformation resembles an amyloidogenic intermediate, it says the protein envisage that induced fit and self-assembly represent complementary molecular adaptations to potential proteotoxicity.
       In order to understand why this change in insulin is complementary to its function you need to understand how insulin works. Insulin is stored as a zinc crystal inside the islet cells in the pancreas, so it has an important function to preserve insulin function. The article explains on page 1 that in pancreatic cells, insulin is stored as Zn2 stabilized hexamers which is shown on page 2 in Fig. 1B. This will form microcrystalline arrays within specialized secretory granules (1). The hexamers will than dissociate upon secretion into the bodies circulation. This will enable the hormone to function as a zinc-free monomer. The monomer is will than undergo a change in conformation upon receptor binding to the cell. This article shows how they investigated a site of conformational change in the B-chain (PheB24) which they tried to show a picture of this folding change in Fig. 1A on page 2 of this article. You see in the normal folding of the crystal structures of insulin, the aromatic side chain anchors an antiparallel B-sheet at the dimer interface which they showed a picture of in Fig. 1C on page 2. Total chemical synthesis is tudied and tested for comparison of corresponding D- and L amino ala amino acid substitutions at this site.

       They do something they call “chiral mutagenesis”. Now I didn’t know what that chiral mutagenesis was when I first read it, but I looked it up on the web, and found an article about it which is named “Chiral mutagenesis of insulin. Foldability and function are inversely regulated by a stereospecific switch in the B chain.” In this article it stated on the first line, “How insulin binds to its receptor is unknown despite decades of investigation. Here, we employ chiral mutagenesis-comparison of corresponding D and L amino acid substitutions in the hormone-to define a structural switch between folding-competent and active conformations.” So they are doing close to the same thing in article I am working on to summarize for you. You can link to this article on chiral mutagenesis of insulin at this link posted below:
http://www.find-health-articles.com/rec_pu...rsely-regulated.htm
I believe it better explains the process they used to make the changes in the insulin, so they could test their hypothesis.

      They use many ways to procedures to study this change in insulin. The consequences of this conformational change are investigated by photomapping of the receptor-binding surface, and you can see in the experimental procedures section of this article they explain how they tested their theory. They ran these tests by using, Synthesis of Insulin Analogs (where human insulin was given by donors), Receptor Binding Assays (where receptor binding was tested), Circular Dichroism—Far-UV CD spectra, 1H NMR Spectroscopy, and Photocross-linking Studies where used to determine the structure of the insulin. Than finally Insulin Fibrillation was used and these tests were performed multiple times.

       The results showed analogs of DKP-insulin containing L-amino acid substitutions at B24 (“L-analogs”) exhibit reduced receptor-binding affinities (relative to the parent monomer), whereas D-analogs exhibit enhanced affinities. No disproportionate changes were observed in the extent of low affinity

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Class: Biochemistry
Professor: Dr. Whalen
Summery report:

Enhancing the Activity of a Protein by Stereospecific Unfolding


Insulin is a protein hormone structure that can be changed by making a substitution of (Ala) where (PheB24) is in the side chain. In this article they claim that the substitution of protein diastereomer D-Ala enhanced the activity of insulin. Insulin has a A chain a larger B chain.

Research Class Report
Professor: Dr. Johnson
Research under Dr. X. Wang

Tuesday Sept. 29th 2009
First day of research Dr. Wang explained his work to us. Testing lean chicken DNA to DNA from a fat chicken, by figure out the differences in DNA. Wanting to find a gap or missing area of the DNA.

Thursday, Oct. 1st 2009
First I worked on the Nano drop ND-1000 SpectrophotometerˇK.I got the results on the amount of DNA from blood extracted from Chicken blood.
We saw how a PCR was run in the lab by the lab assistants.

Tuesday Oct. 6th 2009
Lab Asst TK Fester and I searched for a code to order new primers. Using primer express 2.0 softwareˇKwe pasted a sequence we were told to look for by Dr. Wang and designed primersˇKWe set to software mRNAˇKWhen we found the right 2 codes, we sent primer code off to a lab named Invirogen Illumina

Thursday Oct. 8th 2009
Today I worked on a protein concentrations with Dr. Wang and lab asst. Emenike. Showed how to calculate protein concentrations using the Nanodrop, after doing a western blot and electrolysis jell.

Tuesday Oct 13th 2009
The primers we ordered last week came inˇKWe started mixing the solution in a well plate to run a PCR on the primersˇKAt the end of this day we finished by placing the well plate to be ran in the computer which takes a few hours to run the PCR.

Thursday Oct 15th 2009
Today we extracted DNA from Chicken bloodˇKI wrote all the steps down in my lab book.

Tuesday Oct 20th 2009
Today I did my first PCR and Nanospec drop by myselfˇKI posted the results in my lab note bookˇKOf my 4 samples my 4th sample had the lowest amount of DNA that was isolated from the blood.


Thursday Oct 22nd 2009
We had a meeting today at 10:00 to go over the different research procedures, and see what direction we may go into with the microrayˇK.Today I extracted DNA from Chicken blood, and ran a Nanospec test to see how much DNA I have extracted.



Tuesday Nov. 3rd 2009
Today I went over Microarry software to see how it worksˇKand studied past research done on evaluation of custom microarray applicationsˇKas well as the oligoncleotide design challenge, and a paper on optimized design and assessment of whole genome tiliing arrays. The overall idea of this is to:
„H Identification of differentially expressed genes in the extreme fat chickens and extreme lean chickens
„R Isolate total RNA
„R Reverse transcription and fluorescent labeling
„R Microarray hybridization
„R Data processing and analysis


Thursday Nov. 5th 2009
We had a research meeting with Dr. Wang. We went over our past research experimentsˇKand related science papersˇKtitles areˇKTSPI copy Number Variation Influences Spermatogenesis and shows differences among Y Lineages
We also ran a jell electrophoresis on the DNA samples.

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Edited by author 08-16-2009 11:29 PM
1. You're the Captain of My Sea
2. Sing to Me
3. Call His Name
4. That's Why I Love You
5. Life
6. Signs of God
7. God's Sight
8. Time is Tickin' Away
9. Praise Jesus
10. Follow Me
11. Time to Fly
12. Come Into the Light
13. Calling All God's Children
14. I Can See Clearly Now
15. I Want My Angel Wings
16. Gensis, His 7 Days and 7 Nights
17. Lord Help Me Fix Up My Place
18. Searching Needing you

Sing to me
A melody
With bright green eyes
You’ll change my life
If you just sing to me

Sing to me
A mystery
We’ll have it all
And I just fall
If you sing to me

Sing to me
Sing to me
Sing to me

Sing to me
Alone with me
You’re outta sight
You rock my life
When you sing to me

Sing to me
Of what could be
You’ll be my star
and take me far
If you just sing to me

Sing to me
Sing to me
Sing to me

Sing songs of love and hate
Sing songs of passion and fate

Sing to me
Sing to me
Sing to me

Sing to me
A melody
We’ll have it all
And I just fall
If you sing to me

Sing to me
Sing to me
Sing to me
Sing to me
Sing to me
Sing

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christian music group

Edited by author 04-24-2009 10:25 AM

BMI
609173

I was thirsty and could not see
That you were offering me something
That could quench my thirsting soul
Something that I needed to know
You’ve got water in a living cup
You telling me I should drink up
My little mind must comprehend
The blessing that you’re passing

Taste and See
Drink be free
Blood of Jesus

I was hungry for something more
Than what’s in this earth world
You had some bread I was told
The bread of life for my soul
You said if I just took a bite
My soul would be set right
Hand and hand I’m trusting you
As I bite on though

Taste and See
Eat be free
Bread of Jesus

Why you think
That star it shines
Shining bright
All though the night
Why you think
That ocean’s blue
Did no one
Ever tell you

I think that God made things just the way that they are
To remind people that he is really not so far

Why you think
Why you think

Why you think
That skin your in
Lets you some
Somehow live
Why you think
Those eyes you have
Don’t see that
God’s all is around

Autotrophic Nutrition
  
Autotrophs synthesize organic materials from inorganic materials. Some organisms derive their energy for this process from sunlight and are called photoautotrophs.
  
Example: Green plants
  
Other organisms use chemical energy and are called chemoautotrophs.
  
Example: Nitrifying bacteria. Photoautotrophs are the primary producers in food chains.
  
Heterotrophic Nutrition
Heterotrophs eat ready - made complex organic food. From this they obtain energy for metabolism, atoms and molecules to build new protoplasm or repair worn - out parts, and ions, co-enzymes and vitamins vital for chemical processes. There are four types of heterotrophic nutrition - saprophytic, parasitic, symbiotic and insectivorous.
  
Saprohytic Nutrition (sapros - rotten phyton - plant)

Edited by author 01-26-2009 03:28 AM
Haleluha
Haleluha
haleluha
haleluha
blessed be the name of god
praise to the lord of all
Haleluha
Haleluha
blessed be the name of god
praise to the glorius one
haleluha
haluluha
blessed be the name
blessed be the name
blessed be the name
blessed be the name
haleluha
haleluha
blessed be the name of god
praise to the lord of all
Haleluha
Haleluha
blessed be the name of god
praise to the glorius one
haleluha
haluluha
blessed be the name
blessed be the name
blessed be the name
blessed be the name
(break solo)
haleluha
haleluha
blessed be the name of god
praise to the lord of all
Haleluha
Haleluha
blessed be the name of god
praise to the glorius one
haleluha
haluluha
blessed be the name
blessed be the name
blessed be the name
blessed be the name
haleluha

Edited by author 01-25-2009 10:03 PM
Can't you feel it
feel the freedom
a larger meaning to life
can't you feel it
see the vision
a power that can't be denined

jesus rains jesus saves

can't you feel it
can't you see
can't you feel it
Jesus Saves

can't you feel it
feel his mercy
a bright future after life
can't you feel it
reach out and touch it
shine his light from inside


jesus rains jesus rains
can't you feel it
can't you see
can't you feel it
sweet release


can't you feel it
feel his power
feel your spirit rise
can't you feel it
in your journey
coming close to christ

jesus rains jesus rains
can't you feel it
can't you see
can't you feel it
sweet release

can you feel it
shed your tears and
raise your arms up high
call to Jesus
for the blessing
let your spirit fly

jesus rains jesus rains
can't you feel it
can't you see
can't you feel it
sweet release

hi