Biotechnology Chapter 6

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BiotechnologyChapter 6

• Identifying a Potential Biotechnology Product

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Research and Development of a Potential Product

• What the market and size of the market for this
  product?
•   
• Identify the product source or description of
  procedure.
•  
•  
• Develop a Comprehensive Product Development Plan
  (CPDP)
• Does the product meet a critical need? Who will
  use the product?
•  
•  
• Is the market large enough to produce sufficient
  sales? How many customers are there?
•  
•  
• Is there preliminary data to support that the
  product will work? Will it do what the company
  claims?
• Will it be safe? Without side effects?
•  
•  
•  
• Will there be no patent disputes or can it be
  protected from other companies
•  

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Looking for New Products in Nature

• Other living forms provide many products
• Microbes (gene for round up ready products)
• ...Other Animals (Insulin, skin, bee pollen,
  rennin)
• Plants (Estimates of 1 millions possible
  species)
• Herbal remedies

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Herbal remedies

• Products exhibiting medicinal properties and are
  developed from plants
• Estimated that only 25 of plant species have
  been identified.theres more out there!
• Equatorial rain forests will provide greatest
  number and diversity of unknown

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Examples of Herbals (see table 6.2 pg170)

• Agents Source Condition
• Paclitaxel Yew tree Cancer
• Echinacea Bronchitis, burns, colds, injury,
  leukemia, sinus congestion
• CoQ Heart Disease, obesity,
  periodontis
• Hawthorne berry Angina, heart disease,
  hypertension, sore throat

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Natural Products and Synthetic Equivalent

• Originally from Nature

• Created in a Lab

Soap
Salicin (precursor of aspirin)
Morphine
Sucrose (table sugar)
Penicillin
Insulin from livestock
Detergents
Aspirin
Acetaminophen
Sucralose
Ampicillin
rInsulin (Humalog)
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Herbal remedies

• Chemically, most are compounds that are organic
  aromatic substances.

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Herbal remedies

• Often the effective compound is
• not the actual compound
• but instead
• a secondary
• metabolite.

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Herbal remedies

• In many cases,
• these substances are plant defense mechanisms
• used to protect the plant against predation
  by microorganisms, insects, and herbivores.

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Herbal remedies

• Many of the herbs and spices used by humans to
  season food yield useful medicinal compounds.

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Herbal remedies

• Chemically, most are compounds that are organic
  aromatic substances.
• Often the effective compound is not the actual
  compound, but instead a secondary metabolite.
• In many cases, these substances are plant defense
  mechanisms and used to protect the plant against
  predation by microorganisms, insects, and
  herbivores.
• Many of the herbs and spices used by humans to
  season food yield useful medicinal compounds.

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EXAMPLE Consider the Tannins

• Tannins are astringent, bitter plant polyphenols
• Found in large quantities in the bark of trees
• A barrier for micro-organisms like bacteria and
  fungi and protect the tree.

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Consider Tannins

• Bind and precipitate or shrink proteins.
• The astringency from the tannins is what causes
  the dry and puckery feeling in the mouth
  following the consumption of unripened fruit or
  red wine.

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Phytochemicals provide the starting point for
synthetic products.

• 25 of modern drugs used in the United States
  have been derived from plants.
• Developed by chemists for treatment of various
  diseases

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Used in traditional medicine active
ingredients for prescription drugs

• Among the 120 active compounds currently isolated
  from the higher plants and widely used in modern
  medicine today, 80 percent show a positive
  correlation between their modern therapeutic use
  and the traditional use of the plants from which
  they are derived.

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Provide the starting point for synthetic products.

• More than two thirds of the world's plant species
  - at least 35,000 of which are estimated to have
  medicinal value - come from the developing
  countries.

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Phytochemicals are derivatives of a few
biochemical groups.

• Alkaloids
• Phenolics
• Terpenoids
• Glycosides

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Phytochemicals are derivatives of a few
biochemical groups

• Alkaloids
• contain a
• ring with
• nitrogen

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Similar structure similar effect
Heroine
Acetoaminophen
Morphine
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Phytochemicals are derivatives of a few
biochemical groups

• Caffeine is an alkaloid that provides a mild lift
  
• Many alkaloids
• have dramatic
• effects on the
• central nervous
• system.

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Phytochemicals are derivatives of a few
biochemical groups

• Alkaloids in datura cause severe intoxication and
  even death. (jimson weed, angel's trumpet,
  devil's weed, thorn apple, tolguacha, Jamestown
  weed)

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Phytochemicals are derivatives of a few
biochemical groups

• Alkaloids contain a ring with nitrogen.
• Many alkaloids have dramatic effects on the
  central nervous system.
• Caffeine is an alkaloid that provides a mild lift
  
• Alkaloids in datura cause severe intoxication and
  even death. (jimson weed, angel's trumpet,
  devil's weed, thorn apple, tolguacha, Jamestown
  weed)

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Phytochemicals are derivatives of afew
biochemical groups

• Phenolics contain phenol rings.
• The anthocyanins that give grapes their purple
  color, the isoflavones, the phytoestrogens from
  soy and the tannins that give tea its astringency
  are phenolics

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Phytochemicals are derivatives of afew
biochemical groups

• Phenolics contain
• phenol rings

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Phytochemicals are derivatives of afew
biochemical groups (phenolics)

• The anthocyanins that give grapes their purple
  color, the isoflavones, the phytoestrogens from
  soy and the tannins that give tea its astringency
  are phenolics

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Phytochemicals are derivatives of a few
biochemical groups

• Terpenoids are built up from terpene building
  blocks.
• Each terpene consists of two paired isoprenes.
• Based on the number of isoprene units
• The names monoterpenes, sesquiterpenes,
  diterpenes and triterpene.
• The fragrance of rose and lavender is due to
  monoterpenes.
• The carotenoids produce the reds, yellows and
  oranges of pumpkin, corn and tomatoes.

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Phytochemicals are derivatives of a few
biochemical groups

• Terpenoids are built up from terpene building
  blocks.
• Each terpene consists of two paired isoprenes.
• Isoprene units can be linked together

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Phytochemicals are derivatives of a few
biochemical groups (Terpenoid)

• Based on the number of isoprene units

• The names monoterpenes, sesquiterpenes,
  diterpenes and triterpene.

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Phytochemicals are derivatives of a few
biochemical groups Terpenoids

• Periplanone B, a sesquiterpene (15 carbons), is
  the female sex attractant of a species of
  cockroach. Another sesquiterpene, santonin, is
  also found in wormwood and is a photosensitizer.
  Gossypol is a dimeric sesquiterpene isolated from
  the seeds of cotton plants. It has been used
  clinically in China as a male contraceptive.
• Commonly used ornamental plants, particularly
  when they have a tropical origin, often contain
  biologically active compounds. Many of the
  Euphorbiaceae contain potent skin irritants
  derived from phorbol in their latex. Phorbol, a
  diterpene (20 carbons), is known as a
  cocarcinogen, a compound that is not carcinogenic
  but which promotes carcinogenesis in the presence
  of another agent.

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Phytochemicals are derivatives of a few
biochemical groups Terpenoids

• The fragrance of rose and lavender is due to
  monoterpenes.
• The carotenoids produce the reds, yellows and
  oranges of pumpkin, corn and tomatoes.

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Phytochemicals are derivatives of a few
biochemical groups

• Glycosides consist of a glucose moiety attached
  to an aglycone.
• The aglycone is a molecule that is bioactive in
  its free form but inert until the glycoside bond
  is broken by water or enzymes.
• This mechanism allows the plant to defer the
  availability of the molecule to an appropriate
  time, similar to a safety lock on a gun.
• An example is the cyanoglycosides in cherry pits
  that release toxins only when bitten by a
  herbivore.

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Phytochemicals are derivatives of a few
biochemical groups

• Glycosides consist of a glucose group attached to
  an aglycone.
• The aglycone is a molecule that is bioactive in
  its free form but inert until the bond with sugar
  is broken by water or enzymes.

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Phytochemicals are derivatives of a few
biochemical groups

• This mechanism allows the plant to defer the
  availability of the molecule to an appropriate
  time, similar to a safety lock on a gun.
• alcoholic glycoside is salicin
• Anthraquinone are present in senna, rhubarb and
  aloes they have a laxative effect
• apterin which is reported to dilate the coronary
  arteries as well as block calcium channels
• cyanide group example of these is amygdalin
  from almonds. They can also be found in the
  fruits (and wilting leaves) of the rose family
  (including cherries, apples, plums, almonds,
  peaches, apricots, raspberries, and crabapples

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Phytochemicals are derivatives of a few
biochemical groups

• An example is the cyanoglycosides in cherry pits
  that release toxins only when bitten by a
  herbivore.
• Cyanide is found in amygdalin from almonds Also
  found in fruits (and wilting leaves) of the rose
  family (including cherries, apples, plums,
  almonds, peaches, apricots, raspberries, and
  crabapples
• Alcoholic glycoside is salicin
• Anthraquinone present in senna, rhubarb and
  aloes with a laxative effect
• Apterin which dilates the coronary arteries as
  well as block calcium channels

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Evaluate Coenzyme Q
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Co Enzyme Q

• Claims suggest that it
• Promotes cell growth
• Protects cells from oxidative damage
• Protects body from side effect of cancer therapy
• Stimulates the immune system
• Increases resistance to infections, cancer

• But this is.
• A supplement and not a medicine

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Co enzyme Q

• Also called ubiquinone
• Essential enzyme that functions in the electron
  transport system of mitochondria
• Not regulated by the FDA
• Not proven to be safe or effective

• Pros and cons?

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Supplement vs Medicine and the FDA

• Medicine
• Something that has demonstrated through clinical
  trials (testing) that it treats, prevents, or
  alleviates the symptoms of disease

• Drug
• A chemical that has demonstrated through clinical
  trials that it alters the effects of proteins or
  other molecules associated with a disease-causing
  mechanism

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Phytochemicals can be used to find new
antibiotocs/microbials

• Prevent growth of bacteria or other
  microorganisms by interfering with cellular
  processes
• Important to find new since many bacteria are
  becoming resistant

• Antiseptics are used to
• clean surfaces
• Includes compounds like alcohol, iodine,
  astringents, cleaning agents and toxins

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Testing for Antimicrobials

• Agar dish with a lawn of bacteria
• Soak filter paper discs with agent
• Look for zones of clearing

See table 6.2, page 170
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Testing for Effectiveness

• Products headed for development must be tested
  for
• Activity
• Potency
• Toxicity
• Stability

• Is it present and functioning
• What is the dosage
• Is it toxic to cells, tissue, model organism
• What is the shelf life

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Testing for these characteristics requires an
Assay

• Assay a test
• Many products are proteins
• Need assays for
• Presence
• Activity
• Concentration

• Protein Concentrations using
• Biuret--a copper II) ion s reduced to copper(I),
  which forms a complex with the nitrogens and
  carbons of the peptide bonds in an alkaline
  solution. A violet color indicates the presence
  of proteins
• Bradford, BCA

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Finding Specific Proteins

• Makes use of antibodies
• ELISA Enzyme linked immunosorbent assay
• Western Blot Protein/antibody interaction on a
  membrane

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Assays are required as a product moves through
the pipeline

• When approaching clinical trials---
• Investigational New Drug (IND) application is
  completed

• Pharmacokinetic assays and pharmacodynamic assays
• Amount and length of activity of the protein in
  humans.
• Use animal models first

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NOT ALL Microbes are bad

• Monascus purpureus is an edible fungus imparts a
  distinct flavor and red color when added to
  fermented rice dishes common to Asian food
• the fungus could offer a way to address vitamin A
  deficiency (VAD).

• VAD is a leading cause of preventable blindness
  and increases the risk of illness and death from
  severe infections such as diarrheal disease.

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NOT ALL Microbes are bad

• Geneticist Daniel Skinner replaced the fungus'
  pigment-producing genes with two from another
  species that makes beta-carotene
• Readily converts to vitamin A

Monascus purpureus makes most Asian food very
high in Vit A
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Phenotype vs. Genotype

• We can alter genes to create new traits
• Specific traits phenotype
• Observable expression of genes present phenotype

• Genetic engineering to modify proteins produced
  in an organism

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Biotech's Three Generations

• The first generation refers to the
  herbicide-resistant (Roundup-Ready) and
  biopesticide-producing (Bt) crops, which are now
  planted on tens of millions of acres of farmland.
  

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Biotech's Three Generations

• Generation Two consists of the proposed
  Terminator and Traitor technologies which
  genetically modify plants to produce sterile
  seeds, forcing farmers to buy new seeds each
  year.

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Biotech's Three Generations

• Generation Three consumer-friendly
• GM agricultural plants and farm animals with
  augmented nutritional content, or that produce
  industrial and pharmaceutical chemicals in their
  tissues

• Novel products now being developed include
• antiviral tomato
• rice that produces human proteins for drug
  production
• chickens that produce pharmaceutical drugs in
  their eggs

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Controversy GMOs

• Frankenfoods!!!!!!!!
• Greenpeace is leading a global campaign against
  biotech crops, asserting that they are unhealthy
  and environmentally unsafe

• These groups are adept at sketching out scary
  scenarios. But they have not offered scientific
  evidence that biotech crops have harmed human
  health -- or the natural world.
• http//www.youtube.com/watch?vdsxFZ0rUCoM

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Golden Rice

• International estimates 2 billion people suffer
  from vitamin A deficiency
• about 500,000 children a year go blind from
  vitamin A deficiency
• Most live in developing countries in which rice
  is the dominant grain

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Golden Rice

• Biotech researcher Ingo Potrykus and colleagues
  at the Swiss Federal Institute of Technology
  created a variety of rice that makes
  beta-carotene, the precursor to vitamin A.
• The orange-yellow color of beta-carotene is what
  makes golden rice "golden

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Golden Rice

• Includes three new genes, including two from
  daffodil -- is yellowish and contains
  beta-carotene, a substance that human bodies
  convert to Vitamin A.
• psy (phytoene synthase) from daffodil (Narcissus
  pseudonarcissus)
• crt1 from the soil bacterium Erwinia uredovora
• (The insertion of a lyc (lycopene cyclase) gene
  was thought to be needed but further research
  showed that it is already being produced in
  wild-type rice endosperm.)

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Time Reports in 2000

• The project started in 1992 and at the time of
  publication in 2000, golden rice was considered a
  significant breakthrough in biotechnology as the
  researchers had engineered an entire biosynthetic
  pathway.

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Golden Rice

• There is as yet no GMO rice grown commercially.
• A legal maze of the more than 70 patents that
  are involved in creating golden rice
• Skeptacism on whether it will help malnorished

• Activist groups demanding safety testing
• Studies (2009) at Tufts University and Baylor
  College of medicine safe and effective

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• http//www.youtube.com/watch?vsbxA4WlkUP8

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Recombinant DNA Protein Products

• Discovered in the 1970s
• Discovery of restriction enzymes
• Use of Ligase
• Understanding of gene promotion
• Combine gene for protein of interest with
• regulatory DNA
• Eukaryotic VS Prokaryotic regulation

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Recombinant DNA Protein Products

• E. Coli

• CHO Cells

• Bacteria can take up free DNA in solution
• When taken up by bacteria called TRANSFORMATION
• Expression of gene protein production
• Often use the Lac operon

• Mammalian cells can be COAXED to take up DNA
• When taken up by mammalian cells called
  TRANSFECTION
• Expression of gene protein production
• Use promoter s specific to cells, tissue of choice

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Developing rDNA products

• 15 years average time!
• Preliminary testing
• Potential product ided
• Assays to detect presence and activity
• Determine genetic engineering and production
  methods
• What cells/how to regulate

• Purification of rProtein
• Scale up to manufacture
• Approval from FDA
• Final formulations
• Tablet
• Injectable liquid
• Aerosol inhaler
• Patch
• Cream
• Clinical Trials-3 to 5 yrs

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Product Pipelines

• Every product goes through a product pipeline

• High cost for scientific and non scientific work

• http//www.gene.com/gene/pipeline/status/

• http//media.pfizer.com/files/research/pipeline/20
  09_0331/pipeline_2009_0331.pdf

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Beyond GMOs - Plants are tough

• Dense and woody (remember the Tannins in tree
  bark)
• Grind/grate/mechanically break open cells to get
  at biomolecules
• Cell wall needs to be distupted
• Enzymes Cellulase/Pectinase
• Protoplasts

63
ELISA

• Enzyme-linked immunosorbent assay is a
  biochemical technique used to detect the presence
  of an antibody or an antigen in a sample

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In ELISA..

1. An unknown amount of antigen is affixed to a
   surface
2. Then a specific antibody is washed over the
   surface The antibody binds to the antigen.
3. A secondary antibody, linked to an enzyme, is
   added
4. Chemically developed

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• In the final step a substance is added that the
  enzyme can convert to some detectable signal.

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Fluorescence ELISA

• When light of the appropriate wavelength is shone
  upon the sample, any antigen/antibody complexes
  will fluoresce.
• The amount of antigen in the sample can be
  inferred through the magnitude of the
  fluorescence.

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ELISA FOR DIAGNOSIS

• ELISA can be performed to evaluate either the
  presence of antigen or the presence of antibody
  in a sample
• It is a useful tool for determining serum
  antibody concentrations

• HIV test or West Nile Virus
• It has also found applications in the food
  industry in detecting potential food allergens
• In toxicology as a rapid screen for certain
  classes of drugs.

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ELISA Testing for HIV

• In an ELISA, a person's serum is diluted 400-fold
  and applied to a plate to which HIV antigens are
  attached.
• If antibodies to HIV are present in the serum,
  they may bind to these HIV antigens.

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ELISA Testing for HIV

• The plate is then washed to remove all other
  components of the serum.
• A specially prepared "secondary antibody" an
  antibody that binds to other antibodies is then
  applied to the plate.

• Followed by another wash.
• This secondary antibody is chemically linked in
  advance to an enzyme

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• The plate will contain enzyme in proportion to
  the amount of secondary antibody bound to the
  plate.
• A substrate for the enzyme is applied, and
  catalysis by the enzyme leads to a change in
  color or fluorescence