Purification, Peptide Sequencing and Study of Antiproliferative activity of Laccase against Liver Cancer cell line HepG2 and Human Breast Cancer cell line MCF7
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Three isozymes of laccase were purified from Pleurotus ostreatus strain V-184 using various chromatography techniques. Laccase 1 (LCC 1) was purified to 950-fold and its peptide sequence was deposited in the Uniprot database. LCC 1 exhibited antiproliferative activity against liver cancer Hep G2 and breast cancer MCF 7 cell lines, with IC50 values of 2.8 μM and 3.3 μM respectively. LCC 1 was shown to kill Hep G2 cells through production of hydroxyl radicals via a quinone-redox cycle. A patent was registered for elucidating the mechanism of laccase's anticancer activity. The laccases had optimal temperature
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The document describes the development of a multiplex panel to measure protein levels of HK2, PKM2, and LDHA in frozen tumor biopsies. Antibodies specific to these proteins were coupled to Luminex beads. Recombinant proteins were used to develop sandwich immunoassays and as calibrators. Sample handling procedures generated cytosolic and mitochondrial fractions from tumor tissues. The panel demonstrated proof-of-concept for monitoring PKM2 levels in cancer cell lines treated with PKM2 inhibitors in vitro.
Camara2009 J Bac
This document describes a study characterizing a novel gene cluster involved in the degradation of 4-chlorocatechol by Pseudomonas reinekei MT1. The researchers found that during growth on 5-chlorosalicylate, a novel (chloro)catechol 1,2-dioxygenase (C12OccaA) and a novel (chloro)muconate cycloisomerase (MCIccaB) were induced. MCIccaB was found to transform 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, acting as a functional intermediate between known chloromuconate cycloisomerases and muconate cycloisomerases
JBS 2010
This document describes a study that screened a plant extract library to identify inhibitors of signal transduction pathways mediated by the cholecystokinin receptor CCK1. Researchers developed a beta-lactamase gene reporter assay to detect plant extracts that inhibit CCK1 receptor signaling. They performed high-throughput screening of over 7,000 plant extracts and identified hits using the reporter assay. Researchers then isolated the active compounds from hits and characterized their mechanism of action, identifying a novel selective inhibitor of Gαq/11-coupled receptors.
Molecular docking MAPK.pptx
Best possible natural ligands which were enlisted on NPACT website were screened ( aid of major drug likeness parameters - pkCSM) and docked with the 2OJG(Target protein) using autodock.
Efficient transformation of lactococcus lactis il1403 and generation of knock...
This document describes an optimized protocol for efficiently transforming Lactococcus lactis IL1403 by electroporation. Key aspects of the protocol include growing cells in media supplemented with glycine and sucrose, harvesting them at mid-late log phase, washing them in buffers containing sucrose and EDTA, and electroporating them with a resistor in series. The utility of the protocol was demonstrated by generating single and double gene knock-out mutants using non-replicating vectors. Transformation efficiencies as high as 106 cfu/μg of DNA were achieved, allowing for genetic manipulation of L. lactis IL1403.
IRJET- Subcellular Localization of Transmembrane E-cadherin-GFP Fusion Pr...
1. The study aimed to express an E-cadherin-GFP fusion protein in HeLa cells and examine its subcellular localization. E-cadherin tagged with GFP at its carboxy terminus was cloned into a mammalian expression vector and transfected into HeLa cells.
2. Confocal microscopy of transfected HeLa cells did not show significant GFP signals from the transmembrane region, suggesting the fusion protein was not properly localized to the cell membrane.
3. Quantitative PCR found similar expression levels of the GFP-E-cadherin fusion regardless of whether GFP was on the carboxy or amino terminus, implying tag position did not affect gene expression.
Fluorescence activated cell sorted assay for Gaucher's disease
Gaucher disease results from mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GC) (D-glucosyl-acylsphingosine glucohydrolase, and can be divided into three clinical types on the basis of the presence and severity of neurological involvement. Gaucher disease results from mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GC) (D-glucosyl-acylsphingosine glucohydrolase, and can be divided into three clinical types on the basis of the presence and severity of neurological involvement.
In the case of type 1 Gaucher disease, deficient GC activity leads to an accumulation of the catabolic intermediate glucocerebroside, primarily in macrophages of the reticuloendothelial.
Advances in Capillary Liquid Chromatography for High-Throughput Top Down Prot...
Top Down proteomics has benefited greatly from advances in
mass spectrometry instrumentation and database searching,
yet it has been hindered by the lack of robust separation
platforms for intact proteins. Recently, the use of Gel-Eluted
Liquid Fraction Entrapment Electrophoresis (GELFrEE)1,2
followed by capillary liquid chromatography-MS/MS has
enabled hundreds of Top Down identifications from a single
proteome run.
CcP2APX_Biochem_2008
This document summarizes an experiment to engineer ascorbate peroxidase (APX) activity into cytochrome c peroxidase (CCP) by introducing the APX ascorbate-binding site into CCP. Specifically, the researchers replaced the ascorbate-binding loop and a critical arginine residue in CCP with the corresponding residues from APX to create a mutant called CCP2APX. While wild-type CCP showed no APX activity, CCP2APX was able to catalyze the peroxidation of ascorbate, demonstrating that the engineered ascorbate-binding site could bind ascorbate. Crystal structures of CCP2APX confirmed that the engineered binding site
Aditya - Cell Cycle Article Presentation.pptx
Carnosic acid (CA) is a bioactive molecule found in rosemary and sage plants that has antioxidant and anticancer properties. This study examined the effects of CA on glioblastoma (GBM) cells. The results showed that CA decreased cell survival of GBM cells in a dose-dependent manner by inducing G2 cell cycle arrest and apoptosis. CA modulated key signaling pathways involved in proliferation and survival, including decreasing phosphorylation of STAT3 and AKT. It also promoted proteasomal degradation of proteins important for proliferation like SOX2 and GFAP. However, CA failed to degrade MYC. In conclusion, CA has potential as an anticancer agent for GBM but its clinical potential is limited as it cannot
A family of acetylcholine-gated chloride channel subunits in Caenorhabditis e...
This document summarizes the cloning and characterization of acetylcholine-gated chloride channel subunits in Caenorhabditis elegans. The authors cloned four subunits, ACC-1, ACC-2, ACC-3, and ACC-4, which form a distinct clade of ligand-gated ion channel genes in C. elegans. They found that ACC-1 and ACC-2 form homomeric channels that are gated by acetylcholine but not nicotine, blocked by D-tubocurarine but not α-bungarotoxin, and conduct chloride ions. ACC-3 and ACC-4 may interact with ACC-1 and ACC-2. This study identifies the first molecular
2nd Rotation Report
This document summarizes a study on the heterologous expression and characterization of the c1 dioxygenase enzyme from Tetranychus urticae. Key points:
- The c1 dioxygenase gene was inserted into a plasmid and transformed into E. coli cells for expression. However, initial expression attempts did not show overexpression of the protein.
- Additional expression trials were conducted by varying culture conditions and bacterial clones. SDS-PAGE analysis showed some potential overexpression in new clones induced overnight at 37°C, though further optimization is still needed.
- Once expression is optimized, the goal is to purify the recombinant protein using its His-tag and proceed with structural characterization through crystallization,
Final552 (1)
The document summarizes research analyzing two proteins, AvrGf1 and AvrGf2, that are crucial for the bacterial pathogen Xanthomonas citri to infect citrus plants. Genetic analysis identified the genes for these proteins. Sequence analysis found motifs indicating the proteins are injected into chloroplasts and interact with a citrus cyclophilin after a conformational change. The goal is to express and purify the proteins to study their interaction with the cyclophilin and understand how it activates them during infection.
LCMS PHYTOCHEMICAL ANALYSIS
The document discusses applications of liquid chromatography-mass spectrometry (LCMS). It describes how LCMS combines liquid chromatography separation with mass spectrometry detection. Several studies using LCMS are summarized, including analyzing phytochemicals in Solanum plants and identifying compounds in Archidendron bubalinum seed shell extracts. Key components detected include chlorogenic acids, flavonoids, alkaloids, and the compounds phlorizin and astilbin.
Published Article in PPT.pptx
This is the PowerPoint presentation by extracting data from a research article. This is the title of article" Augmented CO2 tolerance by expressing a single H+-pump enables microalgal valorization of industrial flue gas" published in nature communications. You can download it and ask me to provide you this article as well.
ketoartic_le.pdf
Human aldo-keto reductases (AKR) of the 1A, 1B, 1C and 1D subfamilies are involved in the pre-receptor regulation of nuclear (steroid hormone and orphan) receptors by regulating the local concentrations of their lipophilic ligands. AKR1C3 is one of the most interesting isoforms. It was cloned from human prostate and the recombinant protein was found to function as a 3-, 17- and 20-ketosteroid reductase with a preference for the conversion of Δ4-androstene-3,17- dione to testosterone implicating this enzyme in the local production of active androgens within the prostate. Using a validated isoform specific real-time RT-PCR procedure the AKR1C3 transcript was shown to be more abundant in primary cultures of epithelial cells than stromal cells, and its expression in stromal cells increased with benign and malignant disease. Using a validated isoform specific monoclonal Ab, AKR1C3 protein expression was also detected in prostate epithelial cells by immunoblot analysis.
Immunohistochemical staining of prostate tissue showed that AKR1C3 was expressed in adenocarcinoma and surprisingly high expression was observed in the endothelial cells. These cells are a rich source of prostaglandin G/H synthase 2 (COX-2) and
vasoactive prostaglandins (PG) and thus the ability of recombinant AKR1C enzymes to act as PGF synthases was compared. AKR1C3 had the highest catalytic efficiency (kcat/Km) for the 11-ketoreduction of PGD2 to yield 9α,11β-PGF2 raising the prospect that AKR1C3 may govern ligand access to peroxisome proliferator activated receptor (PPARγ). Activation of PPARγ is often a pro-apoptotic signal and/or leads to terminal differentiation, while 9α,11β- PGF2 is a pro-proliferative signal. AKR1C3 is potently inhibited by non-steroidal anti- inflammatory drugs suggesting that the cancer chemopreventive properties of these agents may be mediated either by inhibition of AKR1C3 or COX. To discriminate between these effects we developed potent AKR1C inhibitors based on N-phenylanthranilic acids that do not inhibit COX-1 or COX-2. These compounds can now be used to determine the role of AKR1C3 in producing two proliferative signals in the prostate namely testosterone and 9α,11β-PGF2.
Optimizing the conjugation of c5 y82f aequorin to the
This document describes the purification of an aequorin protein and its conjugation to an anti-CD33 antibody for potential detection of myeloid leukemia cells. Key steps included transforming E. coli with an aequorin gene, purifying the protein using nickel affinity chromatography, and conjugating the purified aequorin to an anti-CD33 antibody. Initial results showed more aequorin remained unconjugated, but longer incubation times increased luminescence activity though not concentration. Further optimization of conjugation conditions is needed along with future research on detecting CD33 receptors on myeloid leukemia cells.
IN VITRO EVALUATION OF CYTOTOXIC ACTIVITY OF HYDRAZIDEHYDRAZONES BASED ON 4-C...
Two new coumarin-based hydrazide/hydrazones were synthesized and evaluated for their cytotoxicity against two chronic lymphocytic leukemia cell lines. Both compounds showed strong cytotoxicity, with IC50 values several times lower than the reference drug bendamustine. Exposure to the compounds induced fragmentation of leukemic cell nuclei, a hallmark of apoptosis. Quantification assays further confirmed the compounds induced apoptosis in the leukemia cells. The results indicate the new hydrazide/hydrazones have more potent antileukemic effects in vitro compared to bendamustine.
Paloma Pérez-Enfermedades raras de la piel
Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.
2016 RBC RETREAT POSTER TEMPLATE_SMP
- The study characterized the kinetic properties of wild-type human Δ1-pyrroline-5-carboxylate reductase 2 (HsPYCR2) and its R251C mutant found in patients.
- Kinetic analysis showed the R251C mutant had slightly lower catalytic efficiency (4-5 fold) for NADH and NADPH substrates compared to wild-type, suggesting the mutation impacts proline biosynthesis.
- Additional studies on protein structure may help understand how the R251C mutation contributes to neurological disease phenotypes seen in patients.
Similar to Purification, Peptide Sequencing and Study of Antiproliferative activity of Laccase against Liver Cancer cell line HepG2 and Human Breast Cancer cell line MCF7 (20)
Efficient transformation of lactococcus lactis il1403 and generation of knock...
Efficient transformation of lactococcus lactis il1403 and generation of knock...
IRJET- Subcellular Localization of Transmembrane E-cadherin-GFP Fusion Pr...
IRJET- Subcellular Localization of Transmembrane E-cadherin-GFP Fusion Pr...
Fluorescence activated cell sorted assay for Gaucher's disease
Fluorescence activated cell sorted assay for Gaucher's disease
Advances in Capillary Liquid Chromatography for High-Throughput Top Down Prot...
Advances in Capillary Liquid Chromatography for High-Throughput Top Down Prot...
A family of acetylcholine-gated chloride channel subunits in Caenorhabditis e...
A family of acetylcholine-gated chloride channel subunits in Caenorhabditis e...
Optimizing the conjugation of c5 y82f aequorin to the
Optimizing the conjugation of c5 y82f aequorin to the
IN VITRO EVALUATION OF CYTOTOXIC ACTIVITY OF HYDRAZIDEHYDRAZONES BASED ON 4-C...
IN VITRO EVALUATION OF CYTOTOXIC ACTIVITY OF HYDRAZIDEHYDRAZONES BASED ON 4-C...
Purification, Peptide Sequencing and Study of Antiproliferative activity of Laccase against Liver Cancer cell line HepG2 and Human Breast Cancer cell line MCF7
- 1. Purification, Peptide Sequencing and Study of Antiproliferative Activity of Laccase against Liver cancer cell line Hep G2 and human breast cancer cell line MCF 7 Author Antik Kiron Bose See Affilations Senior Scientist and Project Advisor, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Seattle, WA 98109 Summary Three isozymes of laccase LCC 1, LCC 2 and LCC 3 have been purified from Pleurotus ostreatus strain V-184 using Toyopearl DEAE-650 Isoelectric focusing, ConA-sepharose 4B chromatography, Superdex-75 gel filtration, PD10 desalting column and Vydac C-18 Reverse phase HPLC chromatography with 950, 1220 and 935 folds of purification for LCC 1, 2 and 3 respectively. The native molar mass of LCC 1 and 2 heterodimer was calculated to be 130Kda by gel filtration and by 6% SDS-PAGE bands of 65, 60 and 80Kda were obtained for LCC 1, 2 and 3 respectively. Number of SH groups in LCC 1 was calculated to be 4.68 and its full peptide sequencing gave a 533aa protein which was deposited in Protein public database Uniprot with Acc. no. Q12739. IC50 of LCC 1 on Hepatocellular Carcinoma human (Hep G2) and human brest cancer cell lines (MCF 7) were found to be 2.8 and 3.3 µM respectively. 1 to 5µM Menadione with 4.6µM LCC 1 was found to kill Hep G2 cells by production of hydroxyl radical from semiquinone produced by laccase catalysed Quinone-redox cycle. For elucidation of mode of action of laccase on killing Hep G2 cells; a patent has been registered with application no. 3299/DELP/2011 A at CGPDTM, West Bengal. The temperature and pH optima for all isozymes were found to be 30˚C and 5.0 respectively. Spectral analysis showed presence of Type 3 and Type 1 Cu (II) in LCC 1 and reduction of Type 1 during catalysis of Guaiacol. KM and Vmax for 3 isozymes with Guaiacol was found to be 84.034µM, 714.25 U/min; 74.074µM, 204.0816 U/min; 158.73µM, 714.3U/min respectively for LCC 1, 2 and 3. Introduction Laccases, EC 1. 10. 3. 2, p-diphenol: dioxygen oxidoreductase, are part of a larger group of enzymes termed the multi- copper enzymes, which includes among others ascorbic acid oxidase and ceruloplasmin. Laccase was first described by Yoshida (1883) and was characterized as a metal containing oxidase by Bertrand(1985). This makes it one of the oldest enzymes ever described.