Title: Biotechnology Chapter 6
1BiotechnologyChapter 6
- Identifying a Potential Biotechnology Product
2Research 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 -
3Looking 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
4Herbal 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
5Examples 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
6Natural Products and Synthetic Equivalent
Soap
Salicin (precursor of aspirin)
Morphine
Sucrose (table sugar)
Penicillin
Insulin from livestock
Detergents
Aspirin
Acetaminophen
Sucralose
Ampicillin
rInsulin (Humalog)
7Herbal remedies
- Chemically, most are compounds that are organic
aromatic substances.
8Herbal remedies
- Often the effective compound is
- not the actual compound
- but instead
- a secondary
- metabolite.
9Herbal remedies
- In many cases,
- these substances are plant defense mechanisms
- used to protect the plant against predation
by microorganisms, insects, and herbivores.
10Herbal remedies
- Many of the herbs and spices used by humans to
season food yield useful medicinal compounds.
11Herbal 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.
12EXAMPLE 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.
13Consider 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.
14Phytochemicals 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
15Used 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.
16Provide 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.
17Phytochemicals are derivatives of a few
biochemical groups.
- Alkaloids
- Phenolics
- Terpenoids
- Glycosides
18Phytochemicals are derivatives of a few
biochemical groups
- Alkaloids
- contain a
- ring with
- nitrogen
19Similar structure similar effect
Heroine
Acetoaminophen
Morphine
20Phytochemicals 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.
21Phytochemicals 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)
22Phytochemicals 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) -
23Phytochemicals 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
24Phytochemicals are derivatives of afew
biochemical groups
- Phenolics contain
- phenol rings
25Phytochemicals 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
26Phytochemicals 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.
27Phytochemicals 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
28Phytochemicals are derivatives of a few
biochemical groups (Terpenoid)
- Based on the number of isoprene units
- The names monoterpenes, sesquiterpenes,
diterpenes and triterpene.
29Phytochemicals 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.
30Phytochemicals 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.
31Phytochemicals 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.
32Phytochemicals 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.
33Phytochemicals 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
34Phytochemicals 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
35Evaluate Coenzyme Q
36Co 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
37Co 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
38Supplement 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
39Phytochemicals 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
40Testing 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
41Testing 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
42Testing 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
43Finding Specific Proteins
- Makes use of antibodies
- ELISA Enzyme linked immunosorbent assay
- Western Blot Protein/antibody interaction on a
membrane
44Assays 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
45NOT 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.
46NOT 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
47Phenotype 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
48Biotech'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.
49Biotech'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.
50Biotech'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
51Controversy 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
52Golden 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
53Golden 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
54Golden 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.)
55Time 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.
56Golden 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
58Recombinant 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
59Recombinant DNA Protein Products
- 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
60Developing 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
61Product 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
62Beyond 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
63ELISA
- Enzyme-linked immunosorbent assay is a
biochemical technique used to detect the presence
of an antibody or an antigen in a sample
64In ELISA..
- An unknown amount of antigen is affixed to a
surface - Then a specific antibody is washed over the
surface The antibody binds to the antigen. - A secondary antibody, linked to an enzyme, is
added - Chemically developed
65- In the final step a substance is added that the
enzyme can convert to some detectable signal.
66Fluorescence 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.
67ELISA 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.
68ELISA 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.
69ELISA 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