This document summarizes research into the toxicity of the pesticide dieldrin. Experiments showed that dieldrin exposure leads to oxidative stress, mitochondrial dysfunction, and activation of caspase enzymes and pro-apoptotic proteins PKCδ and PARP in dopaminergic neural cells. This triggers cellular apoptosis and DNA fragmentation, demonstrating that dieldrin causes neurodegeneration through inducing programmed cell death pathways.
Light regulated expression of a plant development gene in tomatoArthur Stem
Light is a critical factor for plant development. The importance of this factor has resulted in plants developing a complex signaling system to regulate response to light quality and quantity. Studies of Arabidopsis show that negative regulation is an important component of this process. With this in mind, it’s important to consider the effect light can have on the cessation or continuation of negative regulation found in gene expression. Recently the tomato genome was sequenced; this offers a promising opportunity to study how light regulates gene expression and development in this essential crop. This study looks at the DEETIOLATED-1 (DET1) gene mutation, found in naturally occurring hp2dg (high pigment-2 dark green) tomato plants.
Western Blot Antibody Customer Review for Anti-Endoplasmin Polyclonal Antibod...St John's Laboratory Ltd
The Endoplasmin protein (also known as HSP90B1) is a molecular chaperone encoded by the HSP90B1 gene in humans. It plays a role in the processing and transport of proteins such as integrins and Toll-like receptors. It is located in the endoplasmic reticulum. The Endoplasmin polyclonal antibody detects endogenous levels of Endoplasmin protein.
Light regulated expression of a plant development gene in tomatoArthur Stem
Light is a critical factor for plant development. The importance of this factor has resulted in plants developing a complex signaling system to regulate response to light quality and quantity. Studies of Arabidopsis show that negative regulation is an important component of this process. With this in mind, it’s important to consider the effect light can have on the cessation or continuation of negative regulation found in gene expression. Recently the tomato genome was sequenced; this offers a promising opportunity to study how light regulates gene expression and development in this essential crop. This study looks at the DEETIOLATED-1 (DET1) gene mutation, found in naturally occurring hp2dg (high pigment-2 dark green) tomato plants.
Western Blot Antibody Customer Review for Anti-Endoplasmin Polyclonal Antibod...St John's Laboratory Ltd
The Endoplasmin protein (also known as HSP90B1) is a molecular chaperone encoded by the HSP90B1 gene in humans. It plays a role in the processing and transport of proteins such as integrins and Toll-like receptors. It is located in the endoplasmic reticulum. The Endoplasmin polyclonal antibody detects endogenous levels of Endoplasmin protein.
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
Evaluation of infection course in mice induced by L. major in presence of pos...Nanomedicine Journal (NMJ)
Abstract:
An inoculation of virulent Leishmania major is known as leishmanization (LZ) which is proven to be the most effective control measure against Cutaneous Leishmaniasis (CL). However, using LZ is restricted due to various side effects such as uncontrolled lesion development. In the present research, the efficacy of cationic nanoliposomes containing CpG oligodeoxynucleotides (CpG ODN) as an improved adjuvant delivery system was studied to diminish the lesion development and infection course of L. major after inoculation into the mice. BALB/c mice were inoculated subcutaneously (SC) with L. major plus empty DSPC, DSPC (CpG ODN), DSPC (Non CpG ODN), empty DMPC, DMPC (CpG ODN), DMPC (Non CpG ODN) or HEPES buffer. The results showed that group of mice received DMPC (CpG ODN) nanoliposomes developed a significantly smaller lesion and showed minimum number of L. major in the spleen and draining lymph nodes. In addition, using DMPC (CpG ODN) liposomes resulted in a Th1 type of immune response with a preponderance of IgG2a isotype which is concurrent with the production of DMPC (CpG) induced IFN-γ in the spleen of the mice. Taken together, the results suggested that immune modulation using DMPC (CpG ODN) nanoliposomes might be a practical approach to improve the safety of LZ
Keywords:
DMPC (CpG ODN) nanoliposomes; CpG ODN; L. major; Leishmanization; Immune response
Functions as a master transcriptional regulator of the adaptive response to hypoxia. Under hypoxic conditions, activates the transcription of over 40 genes, including erythropoietin, glucose transporters, glycolytic enzymes, vascular endothelial growth factor, HILPDA, and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia. Plays an essential role in embryonic vascularization, tumor angiogenesis and pathophysiology of ischemic disease. Binds to core DNA sequence 5'-[AG]CGTG-3' within the hypoxia response element (HRE) of target gene promoters. Activation requires recruitment of transcriptional coactivators such as CREBPB and EP300. Activity is enhanced by interaction with both, NCOA1 or NCOA2. Interaction with redox regulatory protein APEX seems to activate CTAD and potentiates activation by NCOA1 and CREBBP. Involved in the axonal distribution and transport of mitochondria in neurons during hypoxia.
Anti-HIF-1α-http://www.stjohnslabs.com/hif-1a-antibody-p-92614
Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation
Austin Virology and Retrovirology is an international scholarly peer reviewed Open Access journal, aims to promote the research in the field of Virology.
Austin Virology and Retrovirology is a comprehensive Open Access peer reviewed scientific Journal that covers multidisciplinary fields. We provide limitless access towards accessing our literature hub with colossal range of articles. The journal aims to publish high quality varied article types such as Research, Review, Case Reports, Short Communications, Perspectives (Editorials), Clinical Images
Austin Virology and Retrovirology supports the scientific modernization and enrichment in virology research community by magnifying access to peer reviewed scientific literary works. Austin also brings universally peer reviewed member journals under one roof thereby promoting knowledge sharing, collaborative and promotion of multidisciplinary science.
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
Evaluation of infection course in mice induced by L. major in presence of pos...Nanomedicine Journal (NMJ)
Abstract:
An inoculation of virulent Leishmania major is known as leishmanization (LZ) which is proven to be the most effective control measure against Cutaneous Leishmaniasis (CL). However, using LZ is restricted due to various side effects such as uncontrolled lesion development. In the present research, the efficacy of cationic nanoliposomes containing CpG oligodeoxynucleotides (CpG ODN) as an improved adjuvant delivery system was studied to diminish the lesion development and infection course of L. major after inoculation into the mice. BALB/c mice were inoculated subcutaneously (SC) with L. major plus empty DSPC, DSPC (CpG ODN), DSPC (Non CpG ODN), empty DMPC, DMPC (CpG ODN), DMPC (Non CpG ODN) or HEPES buffer. The results showed that group of mice received DMPC (CpG ODN) nanoliposomes developed a significantly smaller lesion and showed minimum number of L. major in the spleen and draining lymph nodes. In addition, using DMPC (CpG ODN) liposomes resulted in a Th1 type of immune response with a preponderance of IgG2a isotype which is concurrent with the production of DMPC (CpG) induced IFN-γ in the spleen of the mice. Taken together, the results suggested that immune modulation using DMPC (CpG ODN) nanoliposomes might be a practical approach to improve the safety of LZ
Keywords:
DMPC (CpG ODN) nanoliposomes; CpG ODN; L. major; Leishmanization; Immune response
Functions as a master transcriptional regulator of the adaptive response to hypoxia. Under hypoxic conditions, activates the transcription of over 40 genes, including erythropoietin, glucose transporters, glycolytic enzymes, vascular endothelial growth factor, HILPDA, and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia. Plays an essential role in embryonic vascularization, tumor angiogenesis and pathophysiology of ischemic disease. Binds to core DNA sequence 5'-[AG]CGTG-3' within the hypoxia response element (HRE) of target gene promoters. Activation requires recruitment of transcriptional coactivators such as CREBPB and EP300. Activity is enhanced by interaction with both, NCOA1 or NCOA2. Interaction with redox regulatory protein APEX seems to activate CTAD and potentiates activation by NCOA1 and CREBBP. Involved in the axonal distribution and transport of mitochondria in neurons during hypoxia.
Anti-HIF-1α-http://www.stjohnslabs.com/hif-1a-antibody-p-92614
Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation
Austin Virology and Retrovirology is an international scholarly peer reviewed Open Access journal, aims to promote the research in the field of Virology.
Austin Virology and Retrovirology is a comprehensive Open Access peer reviewed scientific Journal that covers multidisciplinary fields. We provide limitless access towards accessing our literature hub with colossal range of articles. The journal aims to publish high quality varied article types such as Research, Review, Case Reports, Short Communications, Perspectives (Editorials), Clinical Images
Austin Virology and Retrovirology supports the scientific modernization and enrichment in virology research community by magnifying access to peer reviewed scientific literary works. Austin also brings universally peer reviewed member journals under one roof thereby promoting knowledge sharing, collaborative and promotion of multidisciplinary science.
The current slide focuses on different screening models for neurodegenerative diseases along with a brief description of the diseases where the slides are to the points and brief with detailed evaluation.
Neurotoxicity assessment: Comparison between SH-SY5Y and iPSC-derived cellsHCS Pharma
As shown by AstraZeneca in Nature reviews*, one third of the safety failures is linked to CNS toxicity during the clinical trials of drugs. Therefore, relevant in vitro human model is needed to detect early neurotoxicity of drug candidates. In this study, sensitivity of iPSC-derived neuronal cells to 32 compounds is compared to the sensitivity of SH-SY5Y cells. Two types of iPS-derived cells are tested: central nervous system cells (CNS.4U™ cells) and peripheral nervous system cells (PERI.4U™ cells) from Ncardia. Toxic effects are then measured by HCS cell imaging.
This is an internship report on molecular biology techniques, which was performed at PERD center under the guidance of Dr. Anshu Srivastava. This pdf contains all the basic information which is a preliminary requisite to know while approaching the molecular biology experimentally.
PCR (polymerase chain reaction) and Extraction of DNA from fungal plant patho...AjayDesouza V
PCR, Polymerase chain reaction, types of PCR, Template DNA, DNA polymerase, Primers, Nucleotides (DNTPs or deoxynucleotide triphosphates ), Denaturation, Annealing, Extension, Types of PCR, Multiplex PCR.
Long-range PCR.
Single-cell PCR.
Fast-cycling PCR.
Methylation-specific PCR (MSP)
Hot start PCR
High-fidelity PCR.
RAPD: Rapid amplified polymorphic DNA analysis.
Detection of fungal plant pathogen using PCR, Extraction of DNA from plant tissues,PCR amplification and detection of diagnostic amplicon
2. Parkinson’s Disease
Characteristics of
Parkinson’s Disease (PD)
include:
Progressive and selective
dopaminergic neuronal
degeneration
Resting tremor
Bradykinesia
Rigidity
Postural instability
Three different kinds of
PD based on time of onset:
Idiopathic: >40 years old
Young-onset: 21-40 years
old
Juvenile: <20 years old
4. Dieldrin Overview
Formerly known as 1,2,3,4,10,10-hexachloro-6,7-epoxy-
1,4,4a,5,6,7,8,8a-octahydro-end,exo-1,4:5,8-di-
methanonaphthalene (HEOD)
First synthesized in 1946 by Julius Hyman & Co. in
Denver, US
Widely used as an insecticide around the world until
the 1970’s
Was restricted when it was discovered to cause cancer
Officially banned by US EPA in 1987
Classified as one of the top 20 most hazardous
substances to humans by ATSDR
5. Dieldrin Toxicity
Major symptoms of human poisoning include: headache,
nausea, vomiting, convulsion, and coma
Acute lethal dose: 1.5-5 g
Half-life in human blood: 266 days
Dieldrin attacks central nervous system by inhibiting
GABA, leading to hyperexcitation, causing large increase in
Ca2+ levels
Results in: oxidative stress, mitochondrial dysfunction,
activation of caspases, activation of pro-apoptotic signaling
molecules PKCδ and PARP, and cell death via DNA
fragmentation caused by apoptosis
7. Objective
To evaluate the underlying molecular mechanisms
involved with dieldrin-induced dopaminergic
neurodegeneration using:
MTS cell death assay
ROS Assay
Caspase-9 Assay
Caspase-3 Assay
Western blotting of PKCδ, PARP, Bcl2, and Bax
pathways
8. MTS Cell Death Assay
N27 cells were plated in 96-well plate. About 10,000 cells were
plated per well.
Cells were treated with 30μM dieldrin for 3h, 6h, 12h, 18h and
24h.
Post treatment cells were incubated for 90min with 1/5th volume
of MTS dye solution (containing PMS). After incubation, 25μl of
DMSO was added to each well to dissolve the crystals of
formazan.
The absorbance was recorded at 490nm. Also a set of reading
was recorded at wavelength of 670nm as a reference
wavelength in order to eliminate the background.
The final results were converted to % Control and plotted
using GraphPad software. The students t-test was used to
compare the statistical variance. N = 6 were used per treatment
group.
9. MTS Cell Death Assay
C o n tr o l 3 h 6 h 1 2 h 1 8 h 2 4 h
0
5
1 0
1 5
2 0
2 5
3 0
3 5
4 0
4 5
5 0
5 5
6 0
6 5
7 0
7 5
8 0
8 5
9 0
9 5
1 0 0
1 0 5
***
D ie ld rin 3 0 M
CellViability(%Control)
10. ROS Assay
N27 cells were plated in 96-well plate. About 20,000 cells
were plated per well.
Cells were treated with 30μM dieldrin for 3h, 6h, 12h, 18h
and 24h.
Post treatment cells were incubated with 10μM H2-
DCFDA dye for 45min. The experiment was carefully
done in dark after adding the dye.
After dye incubation, the plate was read at em- and ex-
The final results were converted to % Control and plotted
using GraphPad software. The students t-test was used to
compare the statistical variance. N = 6 were used per
treatment group.
11. ROS Assay
C o n tr o l 3 h 6 h 1 2 h 1 8 h 2 4 h
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0
4 0 0
4 5 0
**
***
D ie ld rin 3 0 M
ROSGeneration(%Control)
12. Caspase-9 and -3 activity Assay
N27 cells were plated in 12-well plate. About 100,000 cells were
plated per well.
Cells were treated with 30μM dieldrin for 6h, 12h, 18h and 24h.
Post treatment cells were trypsinized, collected and lysed using
caspase buffer. These lysates were then incubated at 37°C for
20min followed by centrifugation. The supernatant, then, was
incubated with respective caspase substrate at 37°C in a black
well plate.
After 1h of incubation, readings were measured
The caspase values were normalized with mg protein
concentration.
The final results were converted to % Control and plotted
using GraphPad software. The students t-test was used to
compare the statistical variance. N = 4 were used per treatment
group.
13. Caspase-3 activity assay
C o n tr o l 6 h 1 2 h 1 8 h 2 4 h
0
1 0 0
2 0 0
3 0 0
4 0 0
***
D ie ld rin 3 0 M
**
Caspase9Activity(U/mgprotein)
(%Control)
14. Caspase-9 activity assay
C o n tr o l 6 h 1 2 h 1 8 h 2 4 h
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
***
D ie ld rin 3 0 M
*
Caspase3Activity(U/mgprotein)
(%Control)
15. Western Blotting Analysis
N27 cells were plated in T-75 flasks. About 3,000,000 cells
were plated per flask. Cells were treated with 30μM
dieldrin for 3h, 6h, 12h, 18h and 24h.
Post treatment cells harvested and lysed using RIPA buffer
for protein extraction. Protein estimation was done using
Bradford assay.
20μg of protein was added in each well and gels were ran
until the dye passed through the bottom of gels. Proteins
were then transferred on to nitrocellulose membranes.
Post transfer, membranes were blocked for 45min and
then incubated with primary antibody over night.
Followed by secondary antibody incubation for 60min.
Nitrocellulose membranes were developed using LI-COR
fluorescent membrane scanner.
18. Bcl2 and Bax
Bcl-2
(26kDa)
Bax
(23kDa)
DL 30μM
β-actin
- 3h 9h 12h 18h 24h
Gel- 15% Polyacrelamide (2 different gels, one for each)
Primary AB- Rabbit Anti-Bcl-2 (Santa Cruz) 1:1000 overnight
Rabbit Anti-Bax (Santa Cruz) 1:1000 overnight
Mouse Anti-B actin (Sigma) 1:10,000 overnight
Secondary AB- Anti-mouse and Anti-rabbit 1:10,000 for 60min
19. Conclusions
Dieldrin leads to:
Oxidative stress
Mitochondrial dysfunction
Activation of caspases
Activation of pro-apoptotic signaling molecules PKCδ
and PARP
All leading to cell death via DNA fragmentation
caused by apoptosis