scholarly journals Serotonin 5-HT2C Receptor Cys23Ser Single Nucleotide Polymorphism Associates with Receptor Function and Localization In Vitro

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Michelle A. Land ◽  
Holly L. Chapman ◽  
Brionna D. Davis-Reyes ◽  
Daniel E. Felsing ◽  
John A. Allen ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Subcellular Localization ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Calcium Release ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Recycling Pathway

Abstract A non-synonymous single nucleotide polymorphism of the human serotonin 5-HT2C receptor (5-HT2CR) gene that converts a cysteine to a serine at amino acid codon 23 (Cys23Ser) appears to impact 5-HT2CR pharmacology at a cellular and systems level. We hypothesized that the Cys23Ser alters 5-HT2CR intracellular signaling via changes in subcellular localization in vitro. Using cell lines stably expressing the wild-type Cys23 or the Ser23 variant, we show that 5-HT evokes intracellular calcium release with decreased potency and peak response in the Ser23 versus the Cys23 cell lines. Biochemical analyses demonstrated lower Ser23 5-HT2CR plasma membrane localization versus the Cys23 5-HT2CR. Subcellular localization studies demonstrated O-linked glycosylation of the Ser23 variant, but not the wild-type Cys23, may be a post-translational mechanism which alters its localization within the Golgi apparatus. Further, both the Cys23 and Ser23 5-HT2CR are present in the recycling pathway with the Ser23 variant having decreased colocalization with the early endosome versus the Cys23 allele. Agonism of the 5-HT2CR causes the Ser23 variant to exit the recycling pathway with no effect on the Cys23 allele. Taken together, the Ser23 variant exhibits a distinct pharmacological and subcellular localization profile versus the wild-type Cys23 allele, which could impact aspects of receptor pharmacology in individuals expressing the Cys23Ser SNP.

A 3′ UTR Single Nucleotide Polymorphism 829C→T in Dihydrofolate Reductase Gene Results in an Increase in DHFR Protein Level and MTX Resistance.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2084-2084
Author(s):  
Prasunkumar J. Mishra ◽  
Giuseppe S.A. Longo ◽  
Lata G. Menon ◽  
Emine Abali ◽  
Debabrata Banerjee ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Protein Level ◽  
Gene Copy ◽  
Mrna Levels ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Dhfr Gene ◽  
Single Nucleotide

Abstract Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate (THF) required for the synthesis of thymidylate and purines. Methotrexate (MTX) acts as a tight-binding inhibitor of DHFR and remains an important chemotherapeutic agent for treatment of leukemias and lymphomas. Increased DHFR confers resistance to antifolates in target cells. A previously reported single nucleotide polymorphism (SNP) 829C/C→829T/T (829C→T) found in the 3′- untranslated region of DHFR gene transcript (between the first and second polyadenylation site) was associated with higher expression of the DHFR transcript. The SNP was identified in 5.4% of the cases and 6.0% in the controls of Japanese patients with childhood leukemia/lymphomas (Goto et al. 2001, Clinical Cancer Research, Vol. 7, 1952-1956). The objective of the present study was to determine the role of the 3′ UTR SNP 829C→T in DHFR gene expression, DHFR protein level and resistance to MTX. The mutation 829C→T in the 3′ UTR of wild type DHFR was introduced by site directed mutagenesis and the mutant cDNA expressed in DHFR deficient CHO cells (DG-44), wild type DHFR and vector alone constructs were also transfected into DG44 as controls. After two weeks of selection in G418 containing media, several well-isolated surviving colonies were picked and expanded as cell lines in media containing G418. Real-time quantitative PCR was used to compare mRNA and genomic DNA level of the clones while Western blotting was used to compare the protein levels. MTX cytotoxicity assay was carried out in media lacking thymidine. Clones expressing the mutant 829C→T showed greater than two fold enhanced expression of DHFR transcripts as compared to wild type clones. Corresponding to the high mRNA levels, an increase in DHFR protein level was observed in the mutant clones without an increase in DHFR gene copy number. Cytotoxicity studies showed that cell lines with increased levels of DHFR were significantly more resistant to MTX than cells with wild type 3′ UTR. Of interest clonogenic efficiency of the mutants in medium lacking thymidine was greater than wild type and was directly proportional to the level of DHFR expressed in the clones. This study demonstrates that when SNP 829C→T is introduced in the 3′ UTR of wild type DHFR, the expression of the DHFR mRNA is enhanced with a corresponding increase in the protein level. The presence of a SNP 829C→T in patients with ALL may contribute to treatment failure, as MTX is a key drug in curative regimen for this disease. Future studies are directed toward determining the abundance of this SNP in other populations, and the correlation between this SNP and clinical methotrexate resistance and or decreased MTX toxicity.

scholarly journals miR-146a promotes the initiation and progression of melanoma by activating Notch signaling

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Matteo Forloni ◽  
Shaillay Kumar Dogra ◽  
Yuying Dong ◽  
Darryl Conte ◽  
Jianhong Ou ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Notch Signaling ◽  
Cell Lines ◽  
Somatic Mutation ◽  
Melanoma Cell ◽  
Human Melanoma ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Oncogenic Mutations ◽  
Melanoma Cell Lines

Oncogenic mutations in BRAF and NRAS occur in 70% of melanomas. In this study, we identify a microRNA, miR-146a, that is highly upregulated by oncogenic BRAF and NRAS. Expression of miR-146a increases the ability of human melanoma cells to proliferate in culture and form tumors in mice, whereas knockdown of miR-146a has the opposite effects. We show these oncogenic activities are due to miR-146a targeting the NUMB mRNA, a repressor of Notch signaling. Previous studies have shown that pre-miR-146a contains a single nucleotide polymorphism (C>G rs2910164). We find that the ability of pre-miR-146a/G to activate Notch signaling and promote oncogenesis is substantially higher than that of pre-miR-146a/C. Analysis of melanoma cell lines and matched patient samples indicates that during melanoma progression pre-miR-146a/G is enriched relative to pre-miR-146a/C, resulting from a C-to-G somatic mutation in pre-miR-146a/C. Collectively, our results reveal a central role for miR-146a in the initiation and progression of melanoma.

scholarly journals A Novel Single-Nucleotide Polymorphism Loop Mediated Isothermal Amplification Assay for Detection of Artemisinin-Resistant Plasmodium falciparum Malaria

2018 ◽  
Vol 5 (4) ◽  
Author(s):  
Abu Naser Mohon ◽  
Didier Menard ◽  
Mohammad Shafiul Alam ◽  
Kevin Perera ◽  
Dylan R Pillai
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Dynamic Range ◽  
Developed Countries ◽  
Isothermal Amplification ◽  
Test Method ◽  
Nucleotide Polymorphism ◽  
In Vitro Susceptibility ◽  
Single Nucleotide ◽  
Loop Mediated Isothermal Amplification

Abstract Background Artemisinin-resistant malaria (ARM) remains a significant threat to malaria elimination. In the Greater Mekong subregion, the prevalence of ARM in certain regions has reached greater than 90%. Artemisinin-resistant malaria is clinically identified by delayed parasite clearance and has been associated with mutations in the propeller domain of the kelch 13 gene. C580Y is the most prevalent mutation. The detection of ARM currently relies on labor-intensive and time-consuming methods such as clinical phenotyping or in vitro susceptibility testing. Methods We developed a novel single-nucleotide polymorphism loop mediated isothermal amplification (SNP-LAMP) test method for the detection of the C580Y mutation using a novel primer design strategy. Results The SNP-LAMP was 90.0% sensitive (95% confidence interval [CI], 66.9–98.3) and 91.9% specific (95% CI, 82.6–96.7) without knowledge of the parasite load and was 100% sensitive (95% CI, 79.9–100) and 97.3% specific (95% CI, 89.7–99.5) when the parasitemia was within the assay dynamic range. Tests with potential application near-to-patient such as SNP-LAMP may be deployed in low- and middle-income and developed countries. Conclusions Single-nucleotide polymorphism LAMP can serve as a surveillance tool and guide treatment algorithms for ARM in a clinically relevant time frame, prevent unnecessary use of additional drugs that may drive additional resistance, and avoid longer treatment regimens that cause toxicity for the patient.

scholarly journals Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

2018 ◽  
Vol 115 (33) ◽  
pp. 8346-8351 ◽  
Author(s):  
Xiang Li ◽  
Yu Jiang ◽  
Shaorong Chong ◽  
David R. Walt
Keyword(s):  
Single Molecule ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Single Molecule Level ◽  
Subunit Function ◽  
Enzyme Molecules ◽  
Individual Enzyme ◽  
Kinetics Of

In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10−4 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level.

scholarly journals Single-Nucleotide-Polymorphism-Specific PCR for Quantification and Discrimination of Chlamydia pneumoniae Genotypes by Use of a “Locked” Nucleic Acid

2006 ◽  
Vol 72 (5) ◽  
pp. 3785-3787 ◽  
Author(s):  
Jan Rupp ◽  
Werner Solbach ◽  
Jens Gieffers
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Chlamydia Pneumoniae ◽  
Locked Nucleic Acid ◽  
Nucleotide Polymorphisms ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Specific Pcr ◽  
Intraspecies Diversity

ABSTRACT Single-nucleotide polymorphisms (SNPs) are targets to discriminate intraspecies diversity of bacteria and to correlate a genotype with a potential pathotype. Quantification of polygenotypic populations supports this task for in vitro and in vivo applications. We present a novel assay capable of quantifying mixtures of two genotypes differing by only one SNP.

scholarly journals A Novel Assay for Viral MicroRNA Function Identifies a Single Nucleotide Polymorphism That Affects Drosha Processing

2006 ◽  
Vol 80 (11) ◽  
pp. 5321-5326 ◽  
Author(s):  
Eva Gottwein ◽  
Xuezhong Cai ◽  
Bryan R. Cullen
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Target Genes ◽  
Human Herpesvirus ◽  
Northern Analysis ◽  
Luciferase Expression ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Stem Loop

ABSTRACT MicroRNAs (miRNAs) are a class of ∼22-nucleotide noncoding RNAs that inhibit the expression of specific target genes at the posttranscriptional level. Recently, 11 miRNAs encoded by the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) were cloned from latently infected cells. While the expression of these miRNAs has been confirmed by Northern analysis, their ability to inhibit target gene expression has not been demonstrated. We have devised a novel assay for miRNA function that uses lentiviral indicator vectors carrying two perfectly complementary target sites for each given miRNA in the 3′ untranslated region of the Renilla luciferase gene. This assay allowed us to demonstrate the activity of each viral miRNA upon cotransduction of cells with the Renilla luciferase indicator vector together with a firefly luciferase control vector. In KSHV-infected BC-1 and BCBL-1 cells, but not uninfected control cells, Renilla luciferase expression was selectively reduced up to 10-fold. Interestingly, one of the viral miRNAs (miR-K5) exhibited much higher activity in BC-1 cells than in BCBL-1 cells. Sequence analysis of both viral genomes revealed a single nucleotide polymorphism in the miR-K5 precursor stem-loop, which inhibits the expression of mature miR-K5 in BCBL-1 cells. We show that the primary miR-K5 sequence present in BCBL-1 results in diminished processing by Drosha both in vivo and in vitro. This is the first report of a naturally occurring sequence polymorphism in an miRNA precursor that results in reduced processing and therefore lower levels of mature miRNA expression and function.

scholarly journals Novel Genotyping and Quantitative Analysis of Neuraminidase Inhibitor Resistance-Associated Mutations in Influenza A Viruses by Single-Nucleotide Polymorphism Analysis

2011 ◽  
Vol 55 (10) ◽  
pp. 4718-4727 ◽  
Author(s):  
Susu Duan ◽  
David A. Boltz ◽  
Jiang Li ◽  
Christine M. Oshansky ◽  
Henju Marjuki ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Influenza Viruses ◽  
Polymorphism Analysis ◽  
Snp Analysis ◽  
Pandemic H1n1 ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Inhibitor Resistance ◽  
Public Health Epidemiology

ABSTRACTNeuraminidase (NA) inhibitors are among the first line of defense against influenza virus infection. With the increased worldwide use of the drugs, antiviral susceptibility surveillance is increasingly important for effective clinical management and for public health epidemiology. Effective monitoring requires effective resistance detection methods. We have developed and validated a novel genotyping method for rapid detection of established NA inhibitor resistance markers in influenza viruses by single nucleotide polymorphism (SNP) analysis. The multi- or monoplex SNP analysis based on single nucleotide extension assays was developed to detect NA mutations H275Y and I223R/V in pandemic H1N1 viruses, H275Y in seasonal H1N1 viruses, E119V and R292K in seasonal H3N2 viruses, and H275Y and N295S in H5N1 viruses. The SNP analysis demonstrated high sensitivity for low-content NA amplicons (0.1 to 1 ng/μl) and showed 100% accordant results against a panel of defined clinical isolates. The monoplex assays for the H275Y NA mutation allowed precise and accurate quantification of the proportions of wild-type and mutant genotypes in virus mixtures (5% to 10% discrimination), with results comparable to those of pyrosequencing. The SNP analysis revealed the lower growth fitness of an H275Y mutant compared to the wild-type pandemic H1N1 virus by quantitatively genotyping progeny viruses grown in normal human bronchial epithelial cells. This novel method offers high-throughput screening capacity, relatively low costs, and the wide availability of the necessary equipment, and thus it could provide a much-needed approach for genotypic screening of NA inhibitor resistance in influenza viruses.

The role of a single nucleotide polymorphism of MDM2 in glioblastoma multiforme

2008 ◽  
Vol 109 (5) ◽  
pp. 842-848 ◽  
Author(s):  
Rina G. Khatri ◽  
Kapila Navaratne ◽  
Robert J. Weil
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Glioblastoma Multiforme ◽  
Age Of Onset ◽  
Polymerase Chain Reaction Analysis ◽  
Genetic Alterations ◽  
Primary Brain Tumor ◽  
Healthy Controls ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

Object Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a 5-year survival rate of < 5%. Aberrant function of TP53 is common in GBM. Although mutational inactivation of p53 is found in many cases, there remain tumors in which genetic alterations of p53 are absent. Negative regulators of the TP53 pathway such as MDM2, which directly inhibits TP53 expression and activity, may influence the pathogenesis of GBM. To understand its potential function in gliomagenesis, the authors analyzed a novel single nucleotide polymorphism (SNP) in the MDM2 promoter that enhances MDM2 expression. Methods The investigators isolated DNA from 98 patients with GBM and 102 healthy, cancer-free controls. A polymerase chain reaction analysis was performed to determine the MDM2 SNP309 genotype by using distinct primer pairs for the wild-type (T) and mutant (G) alleles. Results The frequency of the mutant MDM2 polymorphism was found to be higher (p = 0.0092) in patients with GBM (54.6%) compared with healthy controls (41.2%); the TT and GG genotypes were more common in healthy controls and patients with GBM (p = 0.0004 and p = 0.02, respectively). Although there was no association between the MDM2 SNP309 and overall survival, the GG genotype was associated with development of GBM at a younger age in patients with tumors harboring wild-type p53, which may mitigate the effect of the MDM2 SNP. Conclusions Although the MDM2 SNP309 does not portend decreased survival, the increased incidence of the mutant G allele in patients with GBM and its influence on age of onset suggest a potential role in the molecular pathogenesis of GBM, and may be a therapeutic target.

Sign in / Sign up

Export Citation Format

Share Document