A presentation on the O'Connor group, given as part of the Advanced Topics of Chemical Biology module. Yet to be awarded a mark, I presented from slide 6 onwards.
Advantages of microbial biotransformation of bioactive compounds & microbial ...Radwa Ahmed
advantages of the use of microbial biotransformation in the field of natural products.
The microbial models for mammalian drug metabolism and applications in drug studies
Important auxiliary method for pathologists in routine diagnostic work as well as in basic and clinical research including exploration of biomarkers, as IHC allows confirmation of target molecule expressions in the context of microenvironment.
pathogenesis, diagnosis, and treatment of diseases are discovered.It is performed without destruction of histologic architecture. It can be used as an effective adjuvant to H & E diagnosis in a majority of tumor cases through the establishment of definitive diagnosis and also gives an insight into tumor histopathogenesis and prognosis.
Molecular Characterization of Isolated Methyl Parathion Degrading Bacteria an...IJSRD
The burning problem in present era is pesticide residue in fruits and foods. present study focus on degradation of pesticide in contest to that total 45 methyl parathion(MP) degrading bacteria were isolated from sludge of MP producing industrial waste. According to screening 21 highly efficient pesticide degraders were selected at 500ppm concentration of standard methyl parathion as a sole carbon source. All isolates were characterized by RAPD and based on the RAPD result all 21 bacterial isolates were grouped in to 2 main clusters with 58% similarity. Characterization of MP degrading gene was carried out by using specific primer. Out of 21 isolates, 14 isolates were containing mpd gene and 4 isolates were containing opd gene while remaining 3 isolates which did not show amplification with opd/ mpd primer so it may contain other unreported gene responsible for methyl parathion degradation. Isolates were identified based on 16s rRNA sequence and one highly efficient bacterium selected for genome characterization.
Cytoprotective and DNA Protective Activity of Carica Papaya Leaf Extractsinventionjournals
Papaya (Carica papaya Linn) is commonly called as paw-paw and it belongs to the family Caricaceae. The properties of papaya fruit and other parts of the plant are also well known in traditional system of medicine. Papaya possess excellent medicinal properties for treatment of different ailments. These curative properties are based on the presence of phytochemical nutrients with antioxidant effect in different parts of the plant. It is considered as valuable nutraceutical fruit plant due to its biological activity and medicinal application.The present study was designed to determine the Cytoprotective and DNA protective activities of different fractions (Aqueous, Chloroform, Ethanol and Ethyl acetate extracts) of Carica papaya leaves. Cytoprotective capacity was assessed using erythrocytes, where ferrous sulphate was used to induce stress and the ability of the extracts to combat the induced stress was evaluated. The DNA protective potential against free radical-mediated oxidative stress was evaluated by a DNA damage inhibition assay involving agarose gel electrophoresis and UV spectrophotometric analysis. All the four fractions displayed significant cytoprotective effect on erythrocytes and prevented oxidative damage to DNA in presence of DNA damaging agent. Altogether, the results of our study lend pharmacological credence to the anti-cancerous and ethno medical use of this plant in traditional system of medicine and these resultscould be used to develop antimutagenic compounds for cancer therapy.
Advantages of microbial biotransformation of bioactive compounds & microbial ...Radwa Ahmed
advantages of the use of microbial biotransformation in the field of natural products.
The microbial models for mammalian drug metabolism and applications in drug studies
Important auxiliary method for pathologists in routine diagnostic work as well as in basic and clinical research including exploration of biomarkers, as IHC allows confirmation of target molecule expressions in the context of microenvironment.
pathogenesis, diagnosis, and treatment of diseases are discovered.It is performed without destruction of histologic architecture. It can be used as an effective adjuvant to H & E diagnosis in a majority of tumor cases through the establishment of definitive diagnosis and also gives an insight into tumor histopathogenesis and prognosis.
Molecular Characterization of Isolated Methyl Parathion Degrading Bacteria an...IJSRD
The burning problem in present era is pesticide residue in fruits and foods. present study focus on degradation of pesticide in contest to that total 45 methyl parathion(MP) degrading bacteria were isolated from sludge of MP producing industrial waste. According to screening 21 highly efficient pesticide degraders were selected at 500ppm concentration of standard methyl parathion as a sole carbon source. All isolates were characterized by RAPD and based on the RAPD result all 21 bacterial isolates were grouped in to 2 main clusters with 58% similarity. Characterization of MP degrading gene was carried out by using specific primer. Out of 21 isolates, 14 isolates were containing mpd gene and 4 isolates were containing opd gene while remaining 3 isolates which did not show amplification with opd/ mpd primer so it may contain other unreported gene responsible for methyl parathion degradation. Isolates were identified based on 16s rRNA sequence and one highly efficient bacterium selected for genome characterization.
Cytoprotective and DNA Protective Activity of Carica Papaya Leaf Extractsinventionjournals
Papaya (Carica papaya Linn) is commonly called as paw-paw and it belongs to the family Caricaceae. The properties of papaya fruit and other parts of the plant are also well known in traditional system of medicine. Papaya possess excellent medicinal properties for treatment of different ailments. These curative properties are based on the presence of phytochemical nutrients with antioxidant effect in different parts of the plant. It is considered as valuable nutraceutical fruit plant due to its biological activity and medicinal application.The present study was designed to determine the Cytoprotective and DNA protective activities of different fractions (Aqueous, Chloroform, Ethanol and Ethyl acetate extracts) of Carica papaya leaves. Cytoprotective capacity was assessed using erythrocytes, where ferrous sulphate was used to induce stress and the ability of the extracts to combat the induced stress was evaluated. The DNA protective potential against free radical-mediated oxidative stress was evaluated by a DNA damage inhibition assay involving agarose gel electrophoresis and UV spectrophotometric analysis. All the four fractions displayed significant cytoprotective effect on erythrocytes and prevented oxidative damage to DNA in presence of DNA damaging agent. Altogether, the results of our study lend pharmacological credence to the anti-cancerous and ethno medical use of this plant in traditional system of medicine and these resultscould be used to develop antimutagenic compounds for cancer therapy.
I. Henderson, J. Ingram, D. Poulcharidis - Advanced Topics in Chemical Biolog...JDIngram
Given as part of the Advanced Topics in Chemical Biology module, Dr Turnbull awarded 75%. I covered slides 6-9, including an animation to explain DNA "toe-hold displacement" when used in catalysed sequential synthesis which was described as "excellent".
This presentation contains b asic information regarding biotechnolgy and genetic engineering required for a food engineer and application of these to food sector.
M Pharm Pharmacognosy Semester 2, MEDICINAL PLANT BIOTECHNOLOGY UNIT 1, Introduction to Plant biotechnology: Historical perspectives, prospects for development of plant biotechnology as a source of
medicinal agents. Applications in pharmacy and allied fields. Genetic and molecular biology as applied to pharmacognosy, study of DNA, RNA and protein replication, genetic code, regulation of gene expression, structure and complicity of
genome, cell signaling, DNA recombinant technology.
genetic engineering, principles, b pharma 6th sem, biotechnology
What is a gene ?
Definition
History
Process
Molecular tools of genetic engineering
Restriction enzymes
History of restriction enzyme
Mechanism of action
Types of restriction enzymes
Application of restriction enzymes
Blunt ends
Sticky ends
transgenic
cisgenic.
knockout organism.
Host organism vector
TRANSGENIC PLANTS
DOLLY THE SHIP
TRANSGENIC ANIMALS
genetic engineering: Genetic engineering, also called genetic modification, is the direct manipulation of an organism's genome using biotechnology. It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms. Many organism are manipulated with the help genetic engineering useful for mankind.
Proteases are protein-degrading enzymes that catalyses hydrolytic reaction in which protein molecules are degraded into peptides and amino acids. Thermostable alkaline proteases are of particular great interest for industrial application because they are stable and active at temperature above 60-70˚C. Thermophiles are found in wide array of environment such as mushroom compost material, nest, hay, wood chips, grains, soil, manure, coal mines etc. Alkaline proteases are most important industrial enzymes and they occupy about 60% of total enzyme market. From the soil samples, eight different fungal species were isolated through soil dilution plate method. In the present study, two fungi Aspergillus nidulans and Aspergillus glaucus from mushroom compost and two fungi Aspergillus terrus, and Aspergillus fumigates from cow manure, showing alkaline protease activity, were isolated. The zones of clearance were observed in Aspergillus nidulans, Aspergillus glaucus, Aspergillus terrus, and Aspergillus fumigatus species of fungi isolated from cow manure and mushroom compost. The best enzyme production was observed in Aspergillus terrus (1.005 ± 0.057 IU/mg protein) obtained from cow manure and the minimum enzyme activity was observed with Aspergillus glaucus (0.278 ± 0.026 IU/mg protein). However, more studies are required to assess the potential of Aspergillus nidulans, Aspergillus glaucus, Aspergillus terrus, and Aspergillus fumigatus species. Key-words- Alkaline protease, Thermophiles, Zone of clearance, Trichloroacetic acid
J. Ingram, D. Poulcharidis - Adv. Topics of Chem. Bio. - Dr. Webb - Prof. S. O'Connor Presentation
1. 1
Advanced Topics of Chemical
Biology
- Dr. M. Webb, University of Leeds
J. Ingram, D. Poulcharidis
- Prof. S. O'Connor
2. 2
The O’Connor Group
Investigates how plants and plant pathogens produce complicated molecules
from simple metabolic building blocks.
• Anti-cancer : Vinblastine and Taxol
• Analgesic: Morphine
• Anti-malarial: Artemisinin and Quinine
Catharanthus roseus
vinblastine
Taxol
Taxol
Taxus brevifolia
Claviceps purpurea
Ergotamine Morphine
Papaver somniferum
3. 3
Areas of Interest
• Metabolic Pathway Elucidation in Medicinal Plants.
• Synthetic Biology with Metabolic Pathways.
• Enzyme Biochemistry, Mechanism and Evolution.
A major focus in the group is to elucidate, understand, and engineer
these pathways so that they can fully harness the wealth of compounds
and biocatalysts that plants have provided.
4. 4
Terpenoid Indole Alkaloids
Are a diverse class of natural products, comprising over 2000 members.
Terpene indole alkaloids are used as anticancer, anti-malarial and anti-arrhythmic agents
All terpene indole alkaloids are derived from tryptophan.
Akuammine tabersonine
Catharanthus roseus
5. 5
Iridoids
Secondary metabolites found in a wide variety of plants and in some animals.
• Intermediates in the biosynthesis of alkaloids
• Consist of a cyclopentane ring fused to a six-membered oxygen heterocycle.
• Found in plants as glycosides.
• Cardiovascular, antihepatotoxic, choleretic, hypoglycemic activities
Iridomyrmex iridomyrmecin
6. Metabolic Pathway Elucidation in 6
Medicinal Plants - Terpene Indole
Alkaloids
• Use bioinformatics to identify potential
genes which code for useful proteins.
• Which genes expressed at known site of
action?
• Test these genes with in vitro assays using
transfection, RNAi and VIGS.
• Goal:
– Improve yield of valuable product/transfect gene
into bacteria/yeast for easier production.
– Adapt new enzymes to expand the library of useful
compounds
7. 7
What is Bioinformatics?
• A powerful tool for
proteomics –
– To use known sequence,
structure, domain or featural
information to predict
features, function or structure
of similar sequence proteins.
8. Identification of a Novel 8
N-Methyltransferase in Vinblastine
Production in Catharanthus roseus
• Knew the stepwise metabolites,
but need to identify an
N-methyltransferase:
• Knew protein was located in
thylakoid membrane.
– Searched the C. roseus genome
for potential methyltransferases
– Select for possible plastidial γ-
tocopherol methyltransferases.
– Select for proteins expressed in
leaves.
S.E. O’Connor et. al. “Homolog of Tocopherol C Methyltransferases Catalyzes N Methylation in Anticancer Alkaloid Biosynthesis”, PNAS, 2010, 107, 44
9. Cytochrome P450 Hydroxylase in 9
Catharanthus roseus:
19-O-acetylhörhammericine production
• Use bioinformatics to identify
proteins likely to interact with
Cytochrome P450
– Discover new proteins involved
in alkaloid biosynthesis.
– Found a unique P450-
dependent enzyme which
stereoselectively hydroxylates
certain alkaloids at a critical
branch point.
S. O’Connor et. al. “Stereoselective Hydroxylation Step of Alkaloid Biosynthesis by a Unique Cytochrome P450 in Caranthus roseus”, J. Bio. Chem, 2011, 286, 19
10. Iridoids – An Alternative Route
to Novel Cyclic Terpenes
O’Connor’s Aims: Uses of Iridoids:
• Identify the • Agricultural Control
reductase protein • Anti-cancer compounds
“Iridoid Synthase”
• Anti-inflammatories
• Explore the effects
of different • Anti-bacterial
reductases on • Anti-fungal Pea aphids,
Acyrthosiphon pisum.
10-oxogeranial
A pest of legume
crops, where
traditional insecticides
kill predators and
parasites.
Geranyl Pyrophosphate 10-Oxogeranial
11. 11
Reductive Cyclisation –
Iridoid Synthase Identification
• Enzyme existence known but not
identified
– Bioinformatics to identify candidates.
– Transfection to E. coli for assays.
– Confirmation and proposed mech.
– Fungal reductase used to produce novel
molecule Inverse electron
demand
Diels–Alder
‡
Michael addition
S.E. O’Connor et. al. “An alternative route to cyclic terpenes by reductive cyclization in iridoid biosynthesis”, Nature, 2012, 138, 494
12. 12
The O’Connor Group:
Jacks of Many Trades
• Explore various complex secondary metabolites
in several different organisms.
– Identify unknown proteins for a valuable product.
– Transfect useful metabolosomes into more viable
hosts.
– Adapt enzymes in various schemes to make novel
non-natural products.
– Bioinformatics to identify related enzymes.