P1888Autoantiboides to Muscarinic M2 cholinoceptors in patients with paroxysmal atrial fibrillation

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
E S Mironova ◽  
N A Mironova ◽  
N Y U Mironov ◽  
L Y U Layovich ◽  
S P Golitsyn ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Amino Acid ◽  
Healthy Subjects ◽  
Holter Monitoring ◽  
Active Role ◽  
Amino Acid Sequences ◽  
Healthy People ◽  
Extracellular Loop ◽  
Autoimmune Processes

Abstract Introduction Atrial fibrillation (AFib) is the most frequenly encountered arrhythmia. In the majority of cases AFib occurs in patients with cardiovascular diseases such as arterial hypertension (AH). In 10–15% of cases AFib occurs in the absense of comorbidities in structurally normal heart. It is reffered to as “lone AFib” and the cause of this arrhythmia remains unknown. Activation of cardiac M2-actylcholinoreceptors (M2-CR) leads to decrease in duration of atrial refractory periods that may contribute to development of AFib. Autoantibodies against M2-CR has cholinomimetic properties, but role of these autoantibodies in development and maintenance of AFib has not been studied. Purpose To assess autoantibodies against M2-CR in patients with paroxysmal lone AFib, in patients with AFib and AH and healthy people. Methods 100 patints with lone Afib, 100 patients with Afib and AH and 25 healthy people were included. Patients underwent clinical blood and urinlysis, assessment of biochemistry blood panel, 12-lead ECG, 24-hour Holter monitoring, echocardiography and stress-testing (treadmill or stress-echocardiography). Assesment of IgM and IgG autoantibodies to M2-CR was performed by indirect immunoenzyme assay. The following peptide molecules were used as epitopes for detection of autoantibodies: M1-amino acid sequence YTVIGYWPLGVVCDL (83–98) of the first extracellular loop of M2-CR; M2-sequence VRTVEDGECYIQFFSNAAVTFGTAI (168–192) of the second extracellular loop of M2-CR; M3-sequence NTFCAPCIPNTV (410–421) of the third extracellular loop of M2-CR, M4-short sequence VEDGECYIQFFS (171–182) of the second extracellular loop of M2-CR; M1+M4-chimeric molecule formed by sequences of the first and the second extacellular loops of M2-CR connected by disulfide bound YTVIGYWPLGVVCDL+VEDGECTIQFFS (83–98+171–182). Results IgG to M2-CR were found in 32% of patients with AFib and in 12% of healthy subjects (p<0,05). IgM to M2-CR were found in 25% of patients with AFib and in 32% of healthy subjects (p<0,05). In patients with lone AFib prevalence of IgG to M2-CR were greater than in patients with AFib and AH (39% vs 25%; p<0,05). Patients with lone AFib and AFib and AH had higher prevalence of IgG to all amino-acid sequences and difference was statistically significant for M2, M4, M1+M4 epitopes (see fig.). Prevalence of IgG to M2-CR epitopes Conclusion Higher prevalence of IgG to M2-CR in patients with AFib than in healthy subjects may suppose an active role of autoimmune processes in arrhythmogenesis.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals Characterization of a Thermoacidophilic l-Arabinose Isomerase from Alicyclobacillus acidocaldarius: Role of Lys-269 in pH Optimum

2005 ◽  
Vol 71 (12) ◽  
pp. 7888-7896 ◽  
Author(s):  
Sang-Jae Lee ◽  
Dong-Woo Lee ◽  
Eun-Ah Choe ◽  
Young-Ho Hong ◽  
Seong-Bo Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Bacillus Halodurans ◽  
Wild Type ◽  
Ph Optimum ◽  
Calculated Molecular Mass ◽  
Alicyclobacillus Acidocaldarius ◽  
Arabinose Isomerase

ABSTRACT The araA gene encoding l-arabinose isomerase (AI) from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius was cloned, sequenced, and expressed in Escherichia coli. Analysis of the sequence revealed that the open reading frame of the araA gene consists of 1,491 bp that encodes a protein of 497 amino acid residues with a calculated molecular mass of 56,043 Da. Comparison of the deduced amino acid sequence of A. acidocaldarius AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to Geobacillus stearothermophilus AI (GSAI) and Bacillus halodurans AI (BHAI), respectively. The recombinant AAAI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The purified enzyme showed maximal activity at pH 6.0 to 6.5 and 65°C under the assay conditions used, and it required divalent cations such as Mn2+, Co2+, and Mg2+ for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent Km values of the recombinant AAAI for l-arabinose and d-galactose were 48.0 mM (V max, 35.5 U/mg) and 129 mM (V max, 7.5 U/mg), respectively, at pH 6 and 65°C. Interestingly, although the biochemical properties of AAAI are quite similar to those of GSAI and BHAI, the three AIs from A. acidocaldarius (pH 6), G. stearothermophilus (pH 7), and B. halodurans (pH 8) exhibited different pH activity profiles. Based on alignment of the amino acid sequences of these homologous AIs, we propose that the Lys-269 residue of AAAI may be responsible for the ability of the enzyme to act at low pH. To verify the role of Lys-269, we prepared the mutants AAAI-K269E and BHAI-E268K by site-directed mutagenesis and compared their kinetic parameters with those of wild-type AIs at various pHs. The pH optima of both AAAI-K269E and BHAI-E268K were rendered by 1.0 units (pH 6 to 7 and 8 to 7, respectively) compared to the wild-type enzymes. In addition, the catalytic efficiency (k cat/Km ) of each mutant at different pHs was significantly affected by an increase or decrease in V max. From these results, we propose that the position corresponding to the Lys-269 residue of AAAI could play an important role in the determination of the pH optima of homologous AIs.

scholarly journals Insights into the mysterious genetic variation profile oftprKinTreponema pallidumunder the development of natural human syphilis infection

2019 ◽  
Author(s):  
Dan Liu ◽  
Man-Li Tong ◽  
Yong Lin ◽  
Li-Li Liu ◽  
Li-Rong Lin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Immune Evasion ◽  
High Frequency ◽  
Critical Role ◽  
Treponema Pallidum ◽  
Secondary Syphilis ◽  
Human Infection ◽  
Amino Acid Sequences ◽  
Potential Vaccine

AbstractAlthough the variations of thetprKgene inTreponema pallidumwere considered to play a critical role in the pathogenesis of syphilis, how actual variable characteristics oftprKin the course of natural human infection enabling the pathogen’s survive has thus far remained unclear. Here, we performed NGS to investigatetprKofT. pallidumdirectly from primary and secondary syphilis samples. Compared with diversity intprKof the strains from primary syphilis samples, there were more mixture variants found within seven V regions of thetprKgene among the strains from secondary syphilis samples, and the frequencies of predominant sequences within V regions oftprKwere generally decreased (less than 80%) with the proportion of minor variants in 10-60% increasing. Noteworthy, the variations within V regions oftprKalways obeyed a strict 3 bp changing pattern. AndtprKin the strains from the two-stage samples kept some stable amino acid sequences within V regions. Particularly, the amino acid sequences IASDGGAIKH and IASEDGSAGNLKH in V1 not only presented a high proportion of inter-population sharing, but also presented a relatively high frequency (above 80%) in the populations. Besides,tprKalways demonstrated remarkable variability in V6 at both the intra- and inter-strain levels regardless of the course. These findings unveiled that the different profile oftprK in T. pallidumdirectly from primary and secondary syphilis samples, indicating that throughout the development of syphilisT. pallidumconstantly varies its domaintprKgene to obtain the best adaptation to the host. While this changing was always subjected a strict gene conversion mechanism to keep an abnormal TprK. The highly stable peptides found in V1 would probably be promising potential vaccine components. And the highly heterogenetic regions (e.g. V6) could provide insight into the mysterious role oftprKin immune evasion.Author summaryAlthough the variations of thetprKgene inTreponema pallidumwere considered to play a critical role in the pathogenesis of syphilis, how actual variable characteristics oftprKin the course of natural human infection enabling the pathogen’s survive has thus far remained unclear. Here, we performed next-generation sequencing, a more sensitive and reliable approach, to investigatetprKofTreponema pallidumdirectly from primary and secondary syphilis patients, revealing that the profile oftprKinT. pallidumfrom the two-stage samples was different. Within the strains from secondary syphilis patients, more mixture variants within seven V regions oftprKwere found, the frequencies of their predominant sequences were generally decreased with the proportion of minor variants in 10-60% was increased. And the variations within V regions oftprKalways obeyed a strict 3 bp changing pattern. Noteworthy, the amino acid sequences IASDGGAIKH and IASEDGSAGNLKH in V1 presented a high proportion of inter-population sharing and presented a relatively high frequency in the populations. And V6 region always demonstrated remarkable variability at intra- and inter-patient levels regardless of the course. These findings provide insights into the mysterious role of TprK in immune evasion and for further exploring the potential vaccine components.

scholarly journals Presence of an isoform of H+-pyrophosphatase located in the alveolar sacs of a scuticociliate parasite of turbot: physiological consequences

Parasitology ◽  
2016 ◽  
Vol 143 (5) ◽  
pp. 576-587 ◽  
Author(s):  
NATALIA MALLO ◽  
JESÚS LAMAS ◽  
ANA-PAULA DEFELIPE ◽  
MARIA-EUGENIA DECASTRO ◽  
ROSA-ANA SUEIRO ◽  
...  
Keyword(s):  
Amino Acid ◽  
Physiological Role ◽  
Amino Acid Sequences ◽  
Cell Structures ◽  
Gene And Protein Expression ◽  
Conserved Gene ◽  
Eukaryotic Organisms ◽  
Cellular Compartments ◽  
First Time

SUMMARYH+-pyrophosphatases (H+-PPases) are integral membrane proteins that couple pyrophosphate energy to an electrochemical gradient across biological membranes and promote the acidification of cellular compartments. Eukaryotic organisms, essentially plants and protozoan parasites, contain various types of H+-PPases associated with vacuoles, plasma membrane and acidic Ca+2storage organelles called acidocalcisomes. We used Lysotracker Red DND-99 staining to identify two acidic cellular compartments in trophozoites of the marine scuticociliate parasitePhilasterides dicentrarchi: the phagocytic vacuoles and the alveolar sacs. The membranes of these compartments also contain H+-PPase, which may promote acidification of these cell structures. We also demonstrated for the first time that theP. dicentrarchiH+-PPase has two isoforms: H+-PPase 1 and 2. Isoform 2, which is probably generated by splicing, is located in the membranes of the alveolar sacs and has an amino acid motif recognized by the H+-PPase-specific antibody PABHK. The amino acid sequences of different isolates of this ciliate are highly conserved. Gene and protein expression in this isoform are significantly regulated by variations in salinity, indicating a possible physiological role of this enzyme and the alveolar sacs in osmoregulation and salt tolerance inP. dicentrarchi.

scholarly journals Role of Transmembrane Domain and Cytoplasmic Tail Amino Acid Sequences of Influenza A Virus Neuraminidase in Raft Association and Virus Budding

2004 ◽  
Vol 78 (10) ◽  
pp. 5258-5269 ◽  
Author(s):  
Subrata Barman ◽  
Lopa Adhikary ◽  
Alok K. Chakrabarti ◽  
Carl Bernas ◽  
Yoshihiro Kawaoka ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Influenza A ◽  
Transmembrane Domain ◽  
Amino Acid Sequences ◽  
Type Ii ◽  
Virus Growth ◽  
Transmembrane Glycoprotein ◽  
Virus Life Cycle

ABSTRACT Influenza virus neuraminidase (NA), a type II transmembrane glycoprotein, possesses receptor-destroying activity and thereby facilitates virus release from the cell surface. Among the influenza A viruses, both the cytoplasmic tail (CT) and transmembrane domain (TMD) amino acid sequences of NA are highly conserved, yet their function(s) in virus biology remains unknown. To investigate the role of amino acid sequences of the CT and TMD on the virus life cycle, we systematically mutagenized the entire CT and TMD of NA by converting two to five contiguous amino acids to alanine. In addition, we also made two chimeric NA by replacing the CT proximal one-third amino acids of the NA TMD [NA(1T2N)NA] and the entire NA TMD (NATRNA) with that of human transferrin receptor (TR) (a type II transmembrane glycoprotein). We rescued transfectant mutant viruses by reverse genetics and examined their phenotypes. Our results show that all mutated and chimeric NAs could be rescued into transfectant viruses. Different mutants showed pleiotropic effects on virus growth and replication. Some mutants (NA2A5, NA3A7, and NA4A10) had little effect on virus growth while others (NA3A2, NA5A27, and NA5A31) produced about 50- to 100-fold-less infectious virus and still some others (NA5A14, NA4A19, and NA4A23) exhibited an intermediate phenotype. In general, mutations towards the ectodomain-proximal sequences of TMD progressively caused reduction in NA enzyme activity, affected lipid raft association, and attenuated virus growth. Electron microscopic analysis showed that these mutant viruses remained aggregated and bound to infected cell surfaces and could be released from the infected cells by bacterial NA treatment. Moreover, viruses containing mutations in the extreme N terminus of the CT (NA3A2) as well as chimeric NA containing the TMD replaced partially [NA(1T2N)NA] or fully (NATRNA) with TR TMD caused reduction in virus growth and exhibited the morphological phenotype of elongated particles. These results show that although the sequences of NA CT and TMD per se are not absolutely essential for the virus life cycle, specific amino acid sequences play a critical role in providing structural stability, enzyme activity, and lipid raft association of NA. In addition, aberrant morphogenesis including elongated particle formation of some mutant viruses indicates the involvement of NA in virus morphogenesis and budding.

scholarly journals Role of the Cytoplasmic Tail Amino Acid Sequences of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein in Virion Incorporation, Cell Fusion, and Pathogenicity

2009 ◽  
Vol 83 (19) ◽  
pp. 10250-10255 ◽  
Author(s):  
Shin-Hee Kim ◽  
Yongqi Yan ◽  
Siba K. Samal
Keyword(s):  
Amino Acid ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Cell Fusion ◽  
Newcastle Disease ◽  
Cytoplasmic Tail ◽  
Amino Acid Sequences ◽  
Amino Acid Deletion ◽  
Virion Incorporation

ABSTRACT To determine the role of amino acid sequences of the hemagglutinin-neuraminidase (HN) cytoplasmic tail in Newcastle disease virus (NDV) replication and pathogenicity, we generated recombinant NDVs with a deletion or point mutation in the N-terminal cytoplasmic tail. The first 2-amino-acid deletion in the cytoplasmic tail did not affect the biological characteristics of NDV. However, a 4-amino-acid deletion and the substitution of alanine for serine at position 6 affected cell fusion, pathogenicity, and colocalization of the HN and M proteins of NDV, indicating that these residues of the HN cytoplasmic tail are critical for its specific incorporation into virions.

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