scholarly journals Reduced pathogenicity of velogenic NDV strain AF22420-I via site-directed mutagenesis of V gene

2021 ◽  
pp. 59-70
Author(s):  
Bei Ru Lee ◽  
Jeevanathan Kalyanasunandram ◽  
Kavitha Murulitharan ◽  
Kok Song Lai ◽  
Suet Lin Chia ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Lethal Dose ◽  
Therapeutic Vaccine ◽  
Premature Stop Codon ◽  
Site Directed Mutagenesis ◽  
Silent Mutation ◽  
V Protein ◽  
P Gene ◽  
Anti Cancer ◽  
Phage T7

Newcastle disease virus (NDV), an avian paramyxovirus, has the potential to be used as an anti-cancer therapeutic vaccine due to its oncolytic and immunostimulatory activities. The virus can be categorised into three pathotypes: lentogenic, mesogenic, and velogenic; of the three pathotypes, the lentogenic strains such as the La Sota are the preferred pathotype for vaccine development due to their low virulence to birds. On the other hand, the translation of the virus to clinic of the velogenic strain AF2240-I is hindered by its virulence towards birds although it exhibits strong oncolysis with significant outcomes both in vitro and in vivo. This study aims to reduce the pathogenicity of AF2240-I yet retaining the anti-cancer properties of the virus. To achieve this, the V protein that acts as an interferon antagonist was chosen to be mutated. It is a non-structural protein that does not interfere with the binding and infection of the virus; hence, mutation of this virulence factor was deducted to be able to reduce harm to the avian species but retain its anti-cancer properties as much as possible. The V protein, which was produced from the insertion of an additional G into a conserved editing site of the P gene, was mutated by substituting the G nucleotide at position 411 from the start of P gene to a T nucleotide. This mutation will produce a premature stop codon from the V mRNA, resulting in a truncated V protein; but only causes a silent mutation in the P protein. The recombinant virus was recovered by the use of BHK cells stably expressing the phage T7 RNA polymerase. The pathogenicity of the mutated virus was determined in 9- to 11-day-old embryonated SPF chicken eggs. The mean death time (MDT) was determined to be 73.6 hours at the minimal lethal dose of 10-7, resembling to that of a mesogenic strain. The virulence of the mutated virus has been successfully reduced where it could be potentially used as the vector for the development of recombinant oncolytic virus for cancer treatment.

scholarly journals Evaluation of Dimer of Epicatechin from an Endophytic Fungus Curvularia australiensis FC2AP on Acute Toxicity Levels, Anti-Inflammatory and Anti-Cervical Cancer Activity in Animal Models

Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 654
Author(s):  
Vellingiri Manon Mani ◽  
Arockiam Jeyasundar Parimala Gnana Soundari ◽  
Balamuralikrishnan Balasubramanian ◽  
Sungkwon Park ◽  
Utthapon Issara ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Acute Toxicity ◽  
Endophytic Fungus ◽  
Lethal Dose ◽  
Chemotherapeutic Agents ◽  
Cancer Drug ◽  
Dependent Manner ◽  
Anti Cancer ◽  
Albino Mice ◽  
Anti Inflammatory

Cervical cancer, as the most frequent cancer in women globally and accounts almost 14% in India. It can be prevented or treated with vaccines, radiation, chemotherapy, and brachytherapy. The chemotherapeutic agents cause adverse post effects by the destruction of the neighboring normal cells or altering the properties of the cells. In order to reduce the severity of the side effects caused by the chemically synthesized therapeutic agents, the current research developed an anti-cancer agent dimer of epicatechin (DoE), a natural bioactive secondary metabolite (BSM) mediated from an endophytic fungus Curvularia australiensis FC2AP. The investigation has initiated with the evaluation of inhibiting the angiogenesis which is a main activity in metastasis, and it was assessed through Hen’s Egg Test on Chorio Allantoic Membrane (HET-CAM) test; the BSM inhibited the growth of blood vessels in the developing chick embryo. Further the DoE was evaluated for its acute toxicity levels in albino mice, whereas the survival dose was found to be 1250 mg/kg and the lethal dose was 1500 mg/kg body weight of albino mice; hematological, biochemical, and histopathological analyses were assessed. The anti-inflammatory responses of the DoE were evaluated in carrageenan induced Wistar rats and the reduction of inflammation occurred in a dose-dependent manner. By fixing the effective dose for anti-inflammation analysis, the DoE was taken for the anti-cervical cancer analysis in benzo (a) pyrene induced female Sprague-Dawley rats for 60 days trial. After the stipulated days, the rats were taken for hematological antioxidants, lipid peroxidation (LPO), member bound enzymes, cervical histopathological and carcinogenic markers analyses. The results specified that the DoE has the capability of reducing the tumor in an efficient way. This is the first report of flavonoid-DoE production from an endophytic fungus C. australiensis has the anticancer potentiality and it can be stated as anti-cancer drug.

scholarly journals A Unique Protein Self-Assembling Nanoparticle with Significant Advantages in Vaccine Development and Production

2020 ◽  
Vol 2020 ◽  
pp. 1-10 ◽  
Author(s):  
Daniel C. Carter ◽  
Brenda Wright ◽  
W. Gray Jerome ◽  
John P. Rose ◽  
Ellen Wilson
Keyword(s):  
Preliminary Analysis ◽  
Vaccine Development ◽  
Regulatory Protein ◽  
Particle Surface ◽  
Therapeutic Vaccine ◽  
Nonstructural Proteins ◽  
Cell Infection ◽  
Self Assembling ◽  
Unique Protein ◽  
Immunogenic Properties

Nanoparticles are playing an increasingly powerful role in vaccine development. Here, we report the repurposing of nonstructural proteins 10 and 11 (hereafter NSP10) from the replicase polyprotein 1a (pp1a) of the human SARS coronavirus (severe acute respiratory syndrome) as a novel self-assembling platform for bioengineered nanoparticles for a variety of applications including vaccines. NSP10 represents a 152 amino acid, 17 kD zinc finger transcription/regulatory protein which self-assembles to form a spherical 84 Å diameter nanoparticle with dodecahedral trigonal 32 point symmetry. As a self-assembling nanoparticle, NSP10 possesses numerous advantages in vaccine development and antigen display, including the unusual particle surface disposition of both the N- and C-termini. Each set of N- or C-termini is spatially disposed in a tetrahedral arrangement and positioned at optimal distances from the 3-fold axes (8-10 Å) to nucleate and stabilize the correct folding of complex helical or fibrous trimeric receptors, such as those responsible for viral tropism and cell infection. An application example in the exploratory development of a therapeutic vaccine for idiopathic pulmonary fibrosis (IPF), including preliminary analysis and immunogenic properties, is presented. The use of this system could accelerate the discovery and development of vaccines for a number of human, livestock, and veterinary applications.

In silico and in vitro analysis of a multiepitope L1-E7 fusion construct for vaccine development against human papillomaviruses

2021 ◽  
Vol 18 ◽  
Author(s):  
Elnaz Abbasifarid ◽  
Azam Bolhassani ◽  
Shiva Irani ◽  
Fattah Sotoodehnejadnematalahi
Keyword(s):  
Western Blotting ◽  
Vaccine Development ◽  
Fluorescent Microscopy ◽  
Therapeutic Vaccine ◽  
Human Papillomaviruses ◽  
Mhc I ◽  
Fusion Construct ◽  
Mhc Ii ◽  
Hpv Types

Background: Human papillomavirus (HPV) infection is the major risk factor for cervical cancer. Current prophylactic HPV vaccines provide immunity against most genital and carcinogenic HPV types. However, these vaccines failed to produce immune responses against already established HPV infections. Methods: For the design of a therapeutic vaccine candidate, we utilized immunoinformatics tools to design a potential multiepitope fusion construct based on L1 and E7 genes from different high- and low-risk HPV types. After determination of CD4+ and CD8+ T cell epitopes, the allergenicity, toxicity, immunogenicity, conservancy, and population coverage were analyzed for epitope selection. Then, the hemolytic probability of the selected epitopes, and molecular docking between major histocompatibility complex (MHC) and the chosen epitopes were performed by different web servers. Next, a multiepitope peptide construct consisting of 12 epitopes linked by the AAY proteasomal sequence was designed. After that, physicochemical properties, solubility, secondary and tertiary structures of this construct were evaluated by bioinformatics tools. Finally, after amino acid reverse translation of the multiepitope peptide construct, expression of the L1-E7 DNA construct (pEGFP-L1-E7) was investigated in HEK-293T cells using fluorescent microscopy, flow cytometry, and western blotting. Results: Considering various parameters, the immunodominant peptides such as L1(MHC-I)-DLDQFPLGRKFLLQ, L1(MHC-II)-NQLFVTVVDTTRSTN, E7-HPV16(MHC-I)-AEPDRAHYNIVTF, E7-HPV18(MHC-I)-HGPKATVQDIVLHL, E7-HPV31(MHC-I)-KPDTSNYNIVTF, E7-HPV33(MHC-I)-RPDGQAQPATADYYI, E7-HPV45(MHC-I)- RTLQQLFLSFV, E7-HPV16(MHC-II)-TLHEYMLDLQPETTD, E7-HPV18(MHC-II)-LRAFQQLFLNTLSFV, E7-HPV31(MHC-II)-PTLQDYVLDLQPEAT, E7-HPV33(MHC-II)-LKEYVLDLYPEPTDL and E7-HPV45(MHC-II)-LQQLFLSTLSFVCPW were determined to design the vaccine construct. The results indicated efficient expression of the L1-E7 DNA construct (74 ± 2.19%) in vitro. Moreover, the polyepitope peptide generated in the cells was detected as a clear band of ~ 50 kDa in western blotting. Conclusion: Regarding the favorable transfection efficiency of the designed L1-E7 multiepitope construct, in vivo validation study on its therapeutic potential is underway.

scholarly journals High-Throughput Identification of New Protective Antigens from a Yersinia pestis Live Vaccine by Enzyme-Linked Immunospot Assay

2009 ◽  
Vol 77 (10) ◽  
pp. 4356-4361 ◽  
Author(s):  
Bei Li ◽  
Lei Zhou ◽  
JingYu Guo ◽  
Xiaoyi Wang ◽  
Bin Ni ◽  
...  
Keyword(s):  
T Cell ◽  
Yersinia Pestis ◽  
Cellular Immunity ◽  
Vaccine Development ◽  
Lethal Dose ◽  
Live Vaccine ◽  
Intracellular Bacteria ◽  
Partial Protection ◽  
Cell Responses ◽  
Protective Antigens

ABSTRACT Yersinia pestis, the plague pathogen, is a facultative intracellular bacterium. Cellular immunity plays important roles in defense against infections. The identification of T-cell targets is critical for the development of effective vaccines against intracellular bacteria; however, the function of cellular immunity in protection from plague was not clearly understood. In this study, 261 genes from Y. pestis were selected on the basis of bioinformatics analysis and previous research results for expression in Escherichia coli BL21(DE3). After purification, 101 proteins were qualified for examination of their abilities to induce the production of gamma interferon in mice immunized with live vaccine EV76 by enzyme-linked immunospot assay. Thirty-four proteins were found to stimulate strong T-cell responses. The protective efficiencies for 24 of them were preliminarily evaluated using a mouse plague model. In addition to LcrV, nine proteins (YPO0606, YPO1914, YPO0612, YPO3119, YPO3047, YPO1377, YPCD1.05c, YPO0420, and YPO3720) may provide partial protection against challenge with a low dose (20 times the 50% lethal dose [20× LD50]) of Y. pestis, but only YPO0606 could partially protect mice from infection with Y. pestis at a higher challenge dosage (200× LD50). These proteins would be the potential components for Y. pestis vaccine development.

scholarly journals Application of Median Lethal Concentration (LC50) of Pathogenic Microorganisms and Their Antigens in Vaccine Development

2020 ◽  
Author(s):  
Saganuwan Alhaji Saganuwan
Keyword(s):  
Staphylococcus Aureus ◽  
Disease Virus ◽  
Vaccine Development ◽  
Hepatitis A Virus ◽  
Lethal Dose ◽  
Vaccine Dose ◽  
Foot And Mouth Disease ◽  
Bacterial Colony ◽  
Mouth Disease Virus

Abstract Objective: Lack of ideal mathematical models to qualify and quantify both pathogenicity, and virulence is a dreadful setback in development of new antimicrobials and vaccines against resistance pathogenic microorganisms.Hence, the modified arithmetical formula of Reed and Muenchhas been integrated with other formulas and used to determine bacterial colony forming unit/ viral concentration, virulence and immunogenicity from LC50s established in the laboratories. Results: Microorganisms’antigens tested are Staphylococcus aureus, Streptococcuspneumonia, Pseudomonas aeruginosa in mice and rat, Edwardsiellaictaluri, Aeromonashydrophila, Aeromonasveronii in fish, New Castle Disease virus in chicken, Sheep Pox Virus, Foot-and-Mouth Disease Virus and Hepatitis A virus in vitro, respectively. The LC50s for the pathogens using different routes of administrations are 1.93 x 103(sheep poxvirus) and 1.75 x 1010 for Staphylococcus aureus (ATCC29213) in rat respectively. Titre index (TI) equals N log10 LC50 and provides protection against lethal dose in graded fashion which translates to protection index. N is the number of vaccine dose that could neutralize the LC50. Hence, parasite inoculum of 103 to 1011could be used as basis for determination of median lethal dose(LD50), LC50 and median bacterial concentrations (BC50)determination, pathogenic dose for immune stimulation should be sought at concentrations less than LC10.

scholarly journals Capsid Assembly is Regulated by Amino Acid Residues Asparagine 47 and 48 in The VP2 Protein of Porcine Parvovirus

2020 ◽  
Author(s):  
Jucai Wang ◽  
Yunchao Liu ◽  
Yumei Chen ◽  
Teng Zhang ◽  
Aiping Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Vaccine Development ◽  
Acid Sites ◽  
Site Directed Mutagenesis ◽  
Porcine Parvovirus ◽  
Capsid Assembly ◽  
Structural Protein ◽  
Native Page ◽  
Vp2 Protein

Abstract Background: Porcine parvovirus (PPV) is a major cause of reproductive failure in swine, and has caused huge losses throughout the world. Viral protein 2 (VP2) of PPV is a major structural protein that can self-assemble into virus-like particles (VLP) with hemagglutination (HA) activity. In order to identify the essential residues involved in the mechanism of capsid assembly and to further understand the function of HA, we analyzed a series of deletion mutants and site-directed mutations within the N-terminal of VP2 in the Escherichia coli (E. coli) system. Results: Our results showed that deletion of first 47 amino acids from the N-terminal of VP2 protein did not affect capsid assembly, and further truncation to residue 48 Asparagine (Asn, N) caused detrimental effects. Site-directed mutagenesis experiments demonstrated that residue 47Asn reduced the assembly efficiency of PPV VLP, while residue 48Asn destroyed the stability, hemagglutination, and self-assembly characteristics of the PPV VP2 protein. These findings indicated that the residues 47Asn and 48Asn are important amino acid sites to capsid assembly and HA activity. Results from Native PAGE inferred that macromolecular polymers were critical intermediates of the VP2 protein during the capsid assembly process. Site-directed mutation at 48Asn did not affect the association of monomers to form into oligomers, but destroyed the ability of oligomers to assemble into macromolecular particles, influencing both capsid assembly and HA activity. Conclusions: These results demonstrated that PPV capsid assembly is a complex process that is regulated by amino acids 47Asn and 48Asn, which are located at the N-terminal of VP2 and closely related to the association of macromolecular particles. Our findings provide valuable information on the mechanisms of PPV capsid assembly and the possibility of chimeric VLP vaccine development by replacing as much as 47 amino acids at the N-terminal of VP2 protein.

scholarly journals Determination of Median Lethal Concentrations (Lc50) of Pathogenic Antigens and Their Applications in Vaccine Development

2020 ◽  
Author(s):  
Saganuwan Alhaji Saganuwan
Keyword(s):  
Staphylococcus Aureus ◽  
Disease Virus ◽  
Vaccine Development ◽  
Hepatitis A Virus ◽  
Lethal Dose ◽  
Vaccine Dose ◽  
Foot And Mouth Disease ◽  
Mouth Disease Virus

Abstract Objective: Lack of ideal mathematical models to qualify and quantify both pathogenicity, and virulence is a dreadful setback in development of new antimicrobials and vaccines against resistance pathogenic microorganisms. Hence, the modified arithmetical formula of Reed and Muench has been integrated with other formulas and used for determination of antigen concentration and parasites inoculums that would kill 50% of test animals (LC50).Results: Microorganisms’ antigens tested are Staphylococcus aureus, Streptococcus pneumonia, Pseudomonas aeruginosa in mice and rat, Edwardsiella ictaluri, Aeromonas hydrophila, Aeromonas veronii in fish, New Castle Disease virus in chicken, Sheep Pox Virus, Foot-and-Mouth Disease Virus and Hepatitis A virus in vitro, respectively. The LC50s for the pathogens using different routes of administrations are 1.93 x 103 (sheep poxvirus) and 1.75 x 1010 for Staphylococcus aureus (ATCC29213) in rat respectively. N is the number of vaccine dose that could neutralize the LC50.Titre index (TI) equals N log10 LC50 and provides protection against lethal dose in graded fashion which translates to protection index. Hence, parasite inoculum of 103 to 1011 could be used as basis for median lethal dose (LD50), LC50 and median bacterial concentrations (BC50) determination, pathogenic dose for immune stimulation should be sought at concentrations less than LC10.

scholarly journals Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy

2021 ◽  
Author(s):  
◽  
Gareth Adrian Prosser
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Alkylating Agents ◽  
Dna Lesions ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
E Coli ◽  
Anti Cancer

<p>Nitroaromatic prodrugs are biologically inert compounds that are attractive candidates for anti-cancer therapy by virtue of their ability to be converted to potent DNA alkylating agents by nitroreductase (NTR) enzymes. In gene-directed enzyme-prodrug therapy (GDEPT), NTR-encoding therapeutic transgenes are delivered specifically to tumour cells, whereupon their expression confers host cell sensitivity to subsequent systemic administration of a nitroaromatic prodrug. The most well studied NTR-GDEPT system involves reduction of the aziridinyl dinitrobenzamide prodrug CB1954 by the Escherichia coli NTR NfsB. However, low affinity of this enzyme for CB1954 has so far limited the clinical efficacy of this GDEPT combination. The research described in this thesis has primarily sought to address this limitation through identification and optimisation of novel NTR enzymes with improved nitroaromatic prodrug reductase activity. Efficient assessment of NTR activity from large libraries of candidate enzymes requires a rapid and reliable screening system. An E. coli-based assay was developed to permit indirect assessment of relative rates of prodrug reduction by over-expressed NTRs via measurement of SOS response induction resulting from reduced prodrug-induced DNA damage. Using this assay in concert with other in vitro and in vivo tests, more than 50 native bacterial NTRs of diverse sequence and origin were assessed for their ability to reduce a panel of clinically attractive nitroaromatic prodrugs. Significantly, a number of NTRs were identified, particularly in the family of enzymes homologous to the native E. coli NTR NfsA, which displayed substantially improved activity over NfsB with CB1954 and other nitroaromatic prodrugs as substrates. This work also examined the roles of E. coli DNA damage repair pathways in processing of adducts induced by various nitroaromatic prodrugs. Of particular interest, nucleotide excision repair was found to be important in the processing of DNA lesions caused by 4-, but not 2-nitro group reduction products of CB1954, which suggests that there are some parallels in the mechanisms of CB1954 adduct repair in E. coli and mammalian cells. Finally, a lead NTR candidate, YcnD from Bacillus subtilis, was selected for further activity improvement through site-directed mutagenesis of active site residues. Using SOS screening, a double-site mutant was identified with 2.5-fold improved activity over the wildtype enzyme in metabolism of the novel dinitrobenzamide mustard prodrug PR-104A. In conclusion, novel NTRs with substantially improved nitroaromatic prodrug reducing activity over previously documented enzymes were identified and characterised. These results hold significance not only for the field of NTR-GDEPT, but also for other biotechnological applications in which NTRs are becoming increasingly significant, including developmental studies, antibiotic discovery and bioremediation. Furthermore, the in vitro assays developed in this study have potential utility in the discovery and evolution of other GDEPT-relevant enzymes whose prodrug metabolism is associated with genotoxicity.</p>

scholarly journals Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy

2021 ◽  
Author(s):  
◽  
Gareth Adrian Prosser
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Alkylating Agents ◽  
Dna Lesions ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
E Coli ◽  
Anti Cancer

<p>Nitroaromatic prodrugs are biologically inert compounds that are attractive candidates for anti-cancer therapy by virtue of their ability to be converted to potent DNA alkylating agents by nitroreductase (NTR) enzymes. In gene-directed enzyme-prodrug therapy (GDEPT), NTR-encoding therapeutic transgenes are delivered specifically to tumour cells, whereupon their expression confers host cell sensitivity to subsequent systemic administration of a nitroaromatic prodrug. The most well studied NTR-GDEPT system involves reduction of the aziridinyl dinitrobenzamide prodrug CB1954 by the Escherichia coli NTR NfsB. However, low affinity of this enzyme for CB1954 has so far limited the clinical efficacy of this GDEPT combination. The research described in this thesis has primarily sought to address this limitation through identification and optimisation of novel NTR enzymes with improved nitroaromatic prodrug reductase activity. Efficient assessment of NTR activity from large libraries of candidate enzymes requires a rapid and reliable screening system. An E. coli-based assay was developed to permit indirect assessment of relative rates of prodrug reduction by over-expressed NTRs via measurement of SOS response induction resulting from reduced prodrug-induced DNA damage. Using this assay in concert with other in vitro and in vivo tests, more than 50 native bacterial NTRs of diverse sequence and origin were assessed for their ability to reduce a panel of clinically attractive nitroaromatic prodrugs. Significantly, a number of NTRs were identified, particularly in the family of enzymes homologous to the native E. coli NTR NfsA, which displayed substantially improved activity over NfsB with CB1954 and other nitroaromatic prodrugs as substrates. This work also examined the roles of E. coli DNA damage repair pathways in processing of adducts induced by various nitroaromatic prodrugs. Of particular interest, nucleotide excision repair was found to be important in the processing of DNA lesions caused by 4-, but not 2-nitro group reduction products of CB1954, which suggests that there are some parallels in the mechanisms of CB1954 adduct repair in E. coli and mammalian cells. Finally, a lead NTR candidate, YcnD from Bacillus subtilis, was selected for further activity improvement through site-directed mutagenesis of active site residues. Using SOS screening, a double-site mutant was identified with 2.5-fold improved activity over the wildtype enzyme in metabolism of the novel dinitrobenzamide mustard prodrug PR-104A. In conclusion, novel NTRs with substantially improved nitroaromatic prodrug reducing activity over previously documented enzymes were identified and characterised. These results hold significance not only for the field of NTR-GDEPT, but also for other biotechnological applications in which NTRs are becoming increasingly significant, including developmental studies, antibiotic discovery and bioremediation. Furthermore, the in vitro assays developed in this study have potential utility in the discovery and evolution of other GDEPT-relevant enzymes whose prodrug metabolism is associated with genotoxicity.</p>

Functional investigation of a highly conserved aspartate residue in the anti -cancer drug efflux transport protein MRP1

2018 ◽  
Author(s):  
Graeme Mullins
Keyword(s):  
Abc Transporters ◽  
Double Mutant ◽  
Transmembrane Helix ◽  
Salt Bridge ◽  
Multidrug Resistant ◽  
Site Directed Mutagenesis ◽  
Cancer Drug ◽  
Wide Range ◽  
Anti Cancer ◽  
Membrane Spanning

Multidrug Resistant Protein 1 (MRP1 or ABCC1) belongs to a subclass of ATP-binding cassette (ABC) transporters that export a wide range of metabolites and xenobiotics across the plasma membrane. Increased expression of MRP1 in cancer cells enhances efflux of many anti-cancer agents, giving rise to multidrug resistant tumours. The purpose of this study was to investigate the function of an aspartate (Asp) amino acid that is highly conserved in all MRP-related proteins by mutating it and determining the consequences of doing so. Asp430 lies at the interface of the cytoplasm and a transmembrane helix in the first membrane-spanning domain of MRP1. Previous studies have shown that when Asp430 is mutated, the protein becomes unstable and is degraded.Because this Asp430 is highly conserved in many MRP-related ABC transporters and because structural homology models of human MRP1 predict that Asp430 is in close proximity to Arg433, we hypothesized that a salt bridge between these two a mino acids could be essential for proper folding and stability of the protein during its biosynthesis. Using site -directed mutagenesis, these two amino acids were interchanged to probe the existence of such an interaction. Thus a double mutant where Asp430 was mutated to Arg, and Arg433 was mutated to Asp was created, and the resultant mutant protein (D430R/R433D) was tested for its ability to be detected in mammalian cells by gel electrophoresis and immunoblotting. Our results show differences between the migration patterns of double and single mutants that are compatible with differences in the glycosylation levels of MRP1. However the fact that D430R and the R433D mutants don’t share the same migration pattern, together with the variation in migration bet ween D430 wild type and Supported by CIHR MOP-10519the double mutant D430R/R433D indicate that the possibility of a salt bridge can be discarded.Supported by CIHR MOP-10519

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