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 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.

Mutation of the β1-tubulin gene associated with congenital macrothrombocytopenia affecting microtubule assembly

Blood ◽  
2009 ◽  
Vol 113 (2) ◽  
pp. 458-461 ◽  
Author(s):  
Shinji Kunishima ◽  
Ryoji Kobayashi ◽  
Tomohiko J. Itoh ◽  
Motohiro Hamaguchi ◽  
Hidehiko Saito
Keyword(s):  
Chinese Hamster ◽  
Microtubule Assembly ◽  
Nucleotide Polymorphism ◽  
Rare Disorders ◽  
Single Nucleotide ◽  
Normal Platelet ◽  
Peripheral Blood Cd34 ◽  
Cd34 Cells ◽  
In Vitro Transfection

Abstract Congenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. We identified the first TUBB1 mutation, R318W, in a patient with congenital macrothrombocytopenia. The patient was heterozygous for Q43P, but this single-nucleotide polymorphism (SNP) did not relate to macrothrombocytopenia. Although no abnormal platelet β1-tubulin localization/marginal band organization was observed, the level of β1-tubulin was decreased by approximately 50% compared with healthy controls. Large and irregular bleb protrusions observed in megakaryocytes derived from the patient's peripheral blood CD34+ cells suggested impaired megakaryocyte fragmentation and release of large platelets. In vitro transfection experiments in Chinese hamster ovary (CHO) cells demonstrated no incorporation of mutant β1-tubulin into microtubules, but the formation of punctuated insoluble aggregates. These results suggested that mutant protein is prone to aggregation but is unstable within megakaryocytes/platelets. Alternatively, mutant β1-tubulin may not be transported from the megakaryocytes into platelets. W318 β1-tubulin may interfere with normal platelet production, resulting in macrothrombocytopenia.

scholarly journals Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution

2020 ◽  
Author(s):  
Ilina Bareja ◽  
Hugo Wioland ◽  
Miro Janco ◽  
Philip R. Nicovich ◽  
Antoine Jégou ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
High Probability ◽  
Actin Binding ◽  
Single Molecule Level ◽  
Filament Dynamics ◽  
Kinetics Of ◽  
Insight Into

ABSTRACTTropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

scholarly journals Codon Substitution Mutations at Two Positions in the L Polymerase Protein of Human Parainfluenza Virus Type 1 Yield Viruses with a Spectrum of Attenuation In Vivo and Increased Phenotypic Stability In Vitro

2004 ◽  
Vol 78 (4) ◽  
pp. 2029-2036 ◽  
Author(s):  
Josephine M. McAuliffe ◽  
Sonja R. Surman ◽  
Jason T. Newman ◽  
Jeffrey M. Riggs ◽  
Peter L. Collins ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nucleotide Substitution ◽  
Wild Type ◽  
Phenotypic Stability ◽  
Single Nucleotide ◽  
Codon Substitution ◽  
Substitution Mutations ◽  
Human Parainfluenza Virus

ABSTRACT The Y942H and L992F temperature-sensitive (ts) and attenuating amino acid substitution mutations, previously identified in the L polymerase of the HPIV3cp45 vaccine candidate, were introduced into homologous positions of the L polymerase of recombinant human parainfluenza virus type 1 (rHPIV1). In rHPIV1, the Y942H mutation specified the ts phenotype in vitro and the attenuation (att) phenotype in hamsters, whereas the L992F mutation specified neither phenotype. Each of these codon mutations was generated by a single nucleotide substitution and therefore had the potential to readily revert to a codon specifying the wild-type amino acid residue. We introduced alternative amino acid assignments at codon 942 or 992 as a strategy to increase genetic stability and to generate mutants that exhibit a range of attenuation. Twenty-three recombinants with codon substitutions at position 942 or 992 of the L protein were viable. One highly ts and att mutant, the Y942A virus, which had a difference of three nucleotides from the codon encoding a wild-type tyrosine, also possessed a high level of genetic and phenotypic stability upon serial passage in vitro at restrictive temperatures compared to that of the parent Y942H virus, which possessed a single nucleotide substitution. We obtained mutants with substitutions at position 992 that, in contrast to the L992F virus, possessed the ts and att phenotypes. These findings identify the use of alternative codon substitution mutations as a method that can be used to generate candidate vaccine viruses with increased genetic stability and/or a modified level of attenuation.

scholarly journals Determination of the Factor V Leiden Single-Nucleotide Polymorphism in a Commercial Clinical Laboratory by Use of NanoChip Microelectronic Array Technology

2002 ◽  
Vol 48 (9) ◽  
pp. 1406-1411 ◽  
Author(s):  
Jess G Evans ◽  
Cindy Lee-Tataseo
Keyword(s):  
Clinical Laboratory ◽  
Factor V Leiden ◽  
Factor V ◽  
Third Wave ◽  
Wild Type ◽  
Wave Analysis ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
The Third ◽  
Array Technology

Abstract Background: Methods for analysis of the single-nucleotide polymorphism (SNP) known as factor V Leiden (FVL) are described. The technique provides rapid, highly accurate detection of the point mutation that encodes for replacement of arginine-506 with glutamine. After formal assay qualification, 758 clinical samples that had previously been analyzed by the InvaderTM Monoplex Assay were tested as research samples in a commercial clinical laboratory. Methods: Primers specific for factor V (FV) were prepared, and PCR was performed. Samples were analyzed using the NanoChip® Molecular Biology Workstation with fluorescently labeled reporters for wild-type and SNP sequences. Results: Of the 635 samples classified by the Third WaveTM assay as FV wild type, 10 were identified as heterozygous FVL by the NanoChip technique. Similarly, of the 114 putative heterozygous samples, 4 were wild type, and of the 9 reported homozygous samples, 6 were homozygous, 2 were heterozygous, and 1 was FV wild type by the NanoChip assay. All 17 results that were discordant with the Third Wave analysis were confirmed by DNA sequencing to be correctly classified by the NanoChip technology. The Nanochip system was 100% accurate in characterizing wild-type, heterozygous, and homozygous samples compared with accuracies of 99.2%, 90.2%, and 100% for the comparable Third Wave analysis. Conclusions: The NanoChip microelectronic chip array technology is an accurate and convenient method for FVL screening of research samples in a clinical laboratory environment.

LSP1 modulates leukocyte populations in resting and inflamed peritoneum

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1827-1835 ◽  
Author(s):  
Jenny Jongstra-Bilen ◽  
Virginia L. Misener ◽  
Chunjie Wang ◽  
Hedy Ginzberg ◽  
Anna Auerbach ◽  
...  
Keyword(s):  
Specific Protein ◽  
Negative Regulator ◽  
Actin Binding ◽  
Wild Type ◽  
Peripheral Tissues ◽  
Lymphocytic Cell ◽  
Peritoneal Mesothelium ◽  
Leukocyte Populations ◽  
Kinetics Of

Abstract Lymphocyte-specific protein 1, recently renamed leukocyte-specific protein 1 (LSP1), is an F-actin binding protein expressed in lymphocytes, macrophages, and neutrophils in mice and humans. This study examines LSP1-deficient (Lsp1−/−) mice for the development of myeloid and lymphocytic cell populations and their response to the development of peritonitis induced by thioglycollate (TG) and to a T-dependent antigen.Lsp1−/− mice exhibit significantly higher levels of resident macrophages in the peritoneum compared to wild-type (wt) mice, whereas the development of myeloid cells is normal. This increase, which is specific for conventional CD5−macrophages appears to be tissue specific and does not result from differences in adhesion to the peritoneal mesothelium. The level of peritoneal lymphocytes is decreased inLsp1−/− mice without affecting a particular lymphocytic subset. The proportions of precursor and mature lymphocytes in the central and peripheral tissues of Lsp1−/−mice are similar to those of wt mice andLsp1−/−mice mount a normal response to the T-dependent antigen, ovalbumin (OVA). On injection of TG, theLsp1−/−mice exhibit an accelerated kinetics of changes in peritoneal macrophage and neutrophil numbers as compared to wt including increased influx of these cells. LSP1− neutrophils demonstrate an enhanced chemotactic response in vitro to N-formyl methionyl-leucyl-phenylalanine (FMLP) and to the C-X-C chemokine, KC, indicating that their enhanced influx into the peritoneum may be a result of increased motility. Our data demonstrate that LSP1 is a negative regulator of neutrophil chemotaxis.

scholarly journals Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets

2019 ◽  
Vol 47 (17) ◽  
pp. e101-e101 ◽  
Author(s):  
Boris Breiner ◽  
Kerr Johnson ◽  
Magdalena Stolarek ◽  
Ana-Luisa Silva ◽  
Aurel Negrea ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Dna Polymerases ◽  
Single Molecule Detection ◽  
Synthesis Methods ◽  
Single Nucleotide ◽  
New Approach ◽  
Fluorescent Signal ◽  
Single Molecule Level ◽  
Current Sequence

AbstractA new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.

scholarly journals Effect of Borrelia burgdorferi OspC at the Site of Inoculation in Mouse Skin

2010 ◽  
Vol 78 (11) ◽  
pp. 4723-4733 ◽  
Author(s):  
Styliani Antonara ◽  
Laura Ristow ◽  
James McCarthy ◽  
Jenifer Coburn
Keyword(s):  
Borrelia Burgdorferi ◽  
Mouse Skin ◽  
Vascular Endothelial ◽  
Wild Type ◽  
Monocyte Chemoattractant Protein 1 ◽  
Monocyte Chemoattractant ◽  
Endothelial Growth Factor ◽  
Kinetics Of ◽  
Precise Role

ABSTRACT The Borrelia burgdorferi surface lipoprotein OspC is a critical virulence factor, but its precise role in the establishment of B. burgdorferi infection remains unclear. To determine whether OspC affects the host response at the site of inoculation of the bacterium, the recruitment of macrophages and neutrophils and the production of cytokines were examined at the site of infection by wild-type, ospC mutant, and complemented mutant B. burgdorferi strains. Of the 21 cytokines tested, monocyte chemoattractant protein 1 (MCP-1), keratinocyte-derived chemokine (KC, CXCL1), and vascular endothelial growth factor (VEGF) were found at increased levels at the site of inoculation of B. burgdorferi, and the levels varied with the production of OspC at one or more time points over the 1-week course of infection. The kinetics of expression and the dependence on OspC production by B. burgdorferi varied among the cytokines. The production of KC and MCP-1, and the appearance of monocytic infiltrates, correlated with the presence of the bacteria rather than with OspC specifically. In contrast, VEGF production was not correlated simply to the presence of the bacteria and is influenced by the presence of OspC. In in vitro assays, OspC and B. burgdorferi expressing OspC stimulated the growth of endothelial cells more than did the controls. These data suggest the possibility of a novel role for OspC in the life of B. burgdorferi at the interface of its mammalian and tick hosts.

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