Biotechnology Chapter 6 - PowerPoint PPT Presentation


Title: Biotechnology Chapter 6


1
BiotechnologyChapter 6
  • Identifying a Potential Biotechnology Product

2
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
  •  

3
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

4
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

5
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

6
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)
7
Herbal remedies
  • Chemically, most are compounds that are organic
    aromatic substances.

8
Herbal remedies
  • Often the effective compound is
  • not the actual compound
  • but instead
  • a secondary
  • metabolite.

9
Herbal remedies
  • In many cases,
  • these substances are plant defense mechanisms
  • used to protect the plant against predation
    by microorganisms, insects, and herbivores.

10
Herbal remedies
  • Many of the herbs and spices used by humans to
    season food yield useful medicinal compounds.

11
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.

12
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.

13
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.

14
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

15
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.

16
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.

17
Phytochemicals are derivatives of a few
biochemical groups.
  • Alkaloids
  • Phenolics
  • Terpenoids
  • Glycosides

18
Phytochemicals are derivatives of a few
biochemical groups
  • Alkaloids
  • contain a
  • ring with
  • nitrogen

19
Similar structure similar effect
Heroine
Acetoaminophen
Morphine
20
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.

21
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)

22
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)

23
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

24
Phytochemicals are derivatives of afew
biochemical groups
  • Phenolics contain
  • phenol rings

25
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

26
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.

27
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

28
Phytochemicals are derivatives of a few
biochemical groups (Terpenoid)
  • Based on the number of isoprene units
  • The names monoterpenes, sesquiterpenes,
    diterpenes and triterpene.

29
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.

30
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.

31
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.

32
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.

33
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

34
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

35
Evaluate Coenzyme Q
36
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

37
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?

38
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

39
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

40
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
41
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

42
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

43
Finding Specific Proteins
  • Makes use of antibodies
  • ELISA Enzyme linked immunosorbent assay
  • Western Blot Protein/antibody interaction on a
    membrane

44
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

45
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.

46
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
47
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

48
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.

49
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.

50
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

51
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

52
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

53
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

54
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.)

55
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.

56
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

57
  • http//www.youtube.com/watch?vsbxA4WlkUP8

58
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

59
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

60
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

61
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

62
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

64
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

65
  • In the final step a substance is added that the
    enzyme can convert to some detectable signal.

66
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.

67
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.

68
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.

69
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

70
  • 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
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Title: Biotechnology Chapter 6


1
BiotechnologyChapter 6
  • Identifying a Potential Biotechnology Product

2
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
  •  

3
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

4
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

5
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

6
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)
7
Herbal remedies
  • Chemically, most are compounds that are organic
    aromatic substances.

8
Herbal remedies
  • Often the effective compound is
  • not the actual compound
  • but instead
  • a secondary
  • metabolite.

9
Herbal remedies
  • In many cases,
  • these substances are plant defense mechanisms
  • used to protect the plant against predation
    by microorganisms, insects, and herbivores.

10
Herbal remedies
  • Many of the herbs and spices used by humans to
    season food yield useful medicinal compounds.

11
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.

12
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.

13
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.

14
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

15
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.

16
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.

17
Phytochemicals are derivatives of a few
biochemical groups.
  • Alkaloids
  • Phenolics
  • Terpenoids
  • Glycosides

18
Phytochemicals are derivatives of a few
biochemical groups
  • Alkaloids
  • contain a
  • ring with
  • nitrogen

19
Similar structure similar effect
Heroine
Acetoaminophen
Morphine
20
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.

21
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)

22
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)

23
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

24
Phytochemicals are derivatives of afew
biochemical groups
  • Phenolics contain
  • phenol rings

25
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

26
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.

27
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

28
Phytochemicals are derivatives of a few
biochemical groups (Terpenoid)
  • Based on the number of isoprene units
  • The names monoterpenes, sesquiterpenes,
    diterpenes and triterpene.

29
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.

30
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.

31
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.

32
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.

33
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

34
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

35
Evaluate Coenzyme Q
36
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

37
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?

38
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

39
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

40
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
41
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

42
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

43
Finding Specific Proteins
  • Makes use of antibodies
  • ELISA Enzyme linked immunosorbent assay
  • Western Blot Protein/antibody interaction on a
    membrane

44
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

45
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.

46
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
47
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

48
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.

49
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.

50
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

51
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

52
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

53
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

54
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.)

55
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.

56
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

57
  • http//www.youtube.com/watch?vsbxA4WlkUP8

58
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

59
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

60
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

61
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

62
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

64
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

65
  • In the final step a substance is added that the
    enzyme can convert to some detectable signal.

66
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.

67
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.

68
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.

69
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

70
  • 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
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