scholarly journals Clinical Features of LMNA-Related Cardiomyopathy in 18 Patients and Characterization of Two Novel Variants

2021 ◽  
Vol 10 (21) ◽  
pp. 5075
Author(s):  
Valentina Ferradini ◽  
Joseph Cosma ◽  
Fabiana Romeo ◽  
Claudia De Masi ◽  
Michela Murdocca ◽  
...  
Keyword(s):  
Heart Disease ◽  
Functional Characterization ◽  
Clinical Manifestations ◽  
Structural Heart Disease ◽  
Left Ventricular ◽  
Dermal Fibroblasts ◽  
Lamin A ◽  
Lmna Gene

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed myocardial performance in the absence of hypertension, valvular, congenital, or ischemic heart disease. Mutations in LMNA gene, encoding for lamin A/C, account for 10% of familial DCM. LMNA-related cardiomyopathies are characterized by heterogeneous clinical manifestations that vary from a predominantly structural heart disease, mainly mild-to-moderate left ventricular (LV) dilatation associated or not with conduction system abnormalities, to highly pro-arrhythmic profiles where sudden cardiac death (SCD) occurs as the first manifestation of disease in an apparently normal heart. In the present study, we select, among 77 DCM families referred to our center for genetic counselling and molecular screening, 15 patient heterozygotes for LMNA variants. Segregation analysis in the relatives evidences other eight heterozygous patients. A genotype–phenotype correlation has been performed for symptomatic subjects. Lastly, we perform in vitro functional characterization of two novel LMNA variants using dermal fibroblasts obtained from three heterozygous patients, evidencing significant differences in terms of lamin expression and nuclear morphology. Due to the high risk of SCD that characterizes patients with lamin A/C cardiomyopathy, genetic testing for LMNA gene variants is highly recommended when there is suspicion of laminopathy.

Abstract 5851: Activation of Inflammatory and Apoptotic Pathways in Human Donor Heart Dysfunction

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Christian F Bulcao ◽  
Karen M D’Souza ◽  
Ricky Malhotra ◽  
Michelle Staron ◽  
Prakash K Pandalai ◽  
...  
Keyword(s):  
Heart Disease ◽  
Structural Heart Disease ◽  
Left Ventricular ◽  
Donor Heart ◽  
Stat3 Pathway ◽  
Stat3 Signaling ◽  
Brain Dead ◽  
Phosphorylated Stat3 ◽  
Proapoptotic Gene

Donor heart dysfunction (DHD) precluding procurement for transplantation occurs in up to 25% of brain dead donors in the absence of structural heart disease. The molecular mechanisms of DHD remain unclear. We investigated the potential role of myocardial interleukin (IL)-6 signaling through the JAK2/STAT3 pathway which leads to the generation of nitric oxide (NO) and decreased cardiac myocyte contractility in vitro. NO can also lead to induction of the proapoptotic gene Bnip3 in vitro. Hearts were procured using standard technique with UW solution from 14 brain dead donors with a left ventricular ejection fraction of < 30% (DHD) in the absence of structural heart disease. Six hearts with normal function (NF) that were procured but not transplanted for non-cardiac reasons served as controls. Left ventricular (LV) IL-6 levels were quantitated by ELISA and signaling through the JAK2/STAT3 pathway via IL-6 and gp130 receptors was assessed by expression of activated, or phosphorylated STAT3. NO signaling was measured by myocardial expression of inducible NO synthase (iNOS) and protein immunoblotting for Bnip3 was performed. Myocardial IL-6 protein levels were 8-fold greater in the DHD group vs. NF controls ( P < 0.02). Phosphorylated STAT3 expression was 5-fold higher in DHD vs. NF ( P < 0.01) indicating increased JAK2/STAT3 signaling as there was no difference in total STAT3 expression between groups ( P >0.05). LV expression of iNOS was 2-fold greater in DHD vs. NF ( P < 0.03) consistent with increased iNOS activity. In addition, LV expression of the proapoptotic gene Bnip3 was 3-fold greater in the DHD group vs. NF ( P < 0.04) suggesting that apoptosis may contribute to DHD. N= 14 for DHD and n= 6 for NF in all studies. Increased myocardial IL-6-mediated signaling through the JAK2/STAT3 pathway leading to upregulation of iNOS and NO production may be an important mechanism in human DHD. Increased myocardial NO also appears to lead to upregulation of proapoptotic Bnip3 in cardiac myocytes, and apoptotic cell death may also contribute to ventricular dysfunction following brain death. Inhibition of IL-6/JAK2/STAT3 signaling may represent a novel strategy to increase the severely limited number of cardiac donors.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals NFB-01. FUNCTIONAL CHARACTERIZATION OF ATRX LOSS IN NF1-ASSOCIATED GLIOMA AND MPNST

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii417-iii418
Author(s):  
Ming Yuan ◽  
Karlyne Reilly ◽  
Christine Pratilas ◽  
Christopher Heaphy ◽  
Fausto Rodriguez
Keyword(s):  
Cell Lines ◽  
Functional Characterization ◽  
Tert Promoter ◽  
Aggressive Tumors ◽  
Sirna Knockdown ◽  
Nih 3T3 ◽  
Vitro Effect ◽  
Murine Glioma

Abstract To identify the biologic relevance of ATRX loss in NF1-associated gliomagenesis, we studied the effects of Atrx loss using four previously characterized Nf1+/-Trp53+/- murine glioma lines. Lines 130G#3 and 158D#8 (corresponding to grade IV and III gliomas, respectively) displayed preserved ATRX protein expression compared to NIH-3T3 cells. We studied the effects of Atrx knockdown in these two lines in the presence and absence of the TERT inhibitor, BIRBR1532. Using a telomere-specific FISH assay, we identified increased signal intensity after Atrx knockdown, only in the presence of the TERT inhibitor. These features are reminiscent of ALT, although there were no significant alterations in cell growth. Next, we studied the effect of ATRX loss in MPNST lines ST88-14, NF90-8, STS-26T. These cell lines all expressed ATRX and DAXX. However, STS-26T contained a TERT promoter mutation and ST88-14 had a known SNP in the TERT promoter, while NF90-8 had no alterations. ATRX siRNA knockdown showed no significant effects in cell proliferation or apoptosis. However, ATRX knockdown resulted in rare ultra-bright foci, indicative of ALT. Next, we studied the in vitro effect of the ATR inhibitor VE-821 in MPNST cell lines. Only NF90-8 (lacking TERT alterations) demonstrated a decrease in growth after ATRX knockdown and VE-821 treatment. However, ATRX knockdown alone did not affect sensitivity to carboplatin. Our findings further support a role for ATRX loss with subsequent ALT activation in a biologic subset of NF1-associated malignancies, thereby opening an opportunity for therapeutic targeting of these aggressive tumors using specific classes of drugs.

Functional Characterization of GABAA Receptor Ligands In Vitro

2004 ◽  
pp. 85-94
Author(s):  
Bjarke Ebert ◽  
Sally Anne Thompson ◽  
Signe Í. Stórustovu ◽  
Keith A. Wafford
Keyword(s):  
Gabaa Receptor ◽  
Functional Characterization ◽  
Receptor Ligands

scholarly journals Biochemical and Functional Characterization of Anthocyanidin Reductase (ANR) from Mangifera indica L.

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2876 ◽  
Author(s):  
Lin Tan ◽  
Mei Wang ◽  
Youfa Kang ◽  
Farrukh Azeem ◽  
Zhaoxi Zhou ◽  
...  
Keyword(s):  
Enzyme Assay ◽  
Molecular Mechanisms ◽  
Mangifera Indica ◽  
Functional Characterization ◽  
Citrate Buffer ◽  
Anthocyanidin Reductase ◽  
High Amino Acid ◽  
Optimum Ph

Mango (Mangifera indica L.) is abundant in proanthocyanidins (PAs) that are important for human health and plant response to abiotic stresses. However, the molecular mechanisms involved in PA biosynthesis still need to be elucidated. Anthocyanidin reductase (ANR) catalyzes a key step in PA biosynthesis. In this study, three ANR cDNAs (MiANR1-1,1-2,1-3) were isolated from mango, and expressed in Escherichia coli. In vitro enzyme assay showed MiANR proteins convert cyanidin to their corresponding flavan-3-ols, such as (−)-catechin and (−)-epicatechin. Despite high amino acid similarity, the recombinant ANR proteins exhibited differences in enzyme kinetics and cosubstrate preference. MiANR1-2 and MiANR1-3 have the same optimum pH of 4.0 in citrate buffer, while the optimum pH for MiANR1-1 is pH 3.0 in phosphate buffer. MiANR1-1 does not use either NADPH or NADH as co-substrate while MiANR1-2/1-3 use only NADPH as co-substrate. MiANR1-2 has the highest Km and Vmax for cyanidin, followed by MiANR1-3 and MiANR1-1. The overexpression of MiANRs in ban mutant reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate MiANRs can form the ANR pathway, leading to the formation of two types of isomeric flavan-3-ols and PAs in mango.

scholarly journals N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Stanislav Huszár ◽  
Vinayak Singh ◽  
Alica Polčicová ◽  
Peter Baráth ◽  
María Belén Barrio ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Functional Characterization ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Libraries ◽  
Whole Cells ◽  
Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

scholarly journals Antiarrhythmic Medication for Atrial Fibrillation (AIM-AF) study: A physician survey of antiarrhythmic drug (AAD) treatment practices and guideline adherence in the EU and USA

EP Europace ◽  
2021 ◽  
Vol 23 (Supplement_3) ◽  
Author(s):  
AJ Camm ◽  
C Blomstrom-Lundqvist ◽  
G Boriani ◽  
A Goette ◽  
PR Kowey ◽  
...  
Keyword(s):  
Heart Disease ◽  
Structural Heart Disease ◽  
Left Ventricular ◽  
Rhythm Control ◽  
Left Ventricular Ejection ◽  
Primary Therapy ◽  
Physician Survey ◽  
Treatment Practices ◽  
The Usa ◽  
The Eu

Abstract Funding Acknowledgements Type of funding sources: Private company. Main funding source(s): Sanofi Introduction The 2020 European Society of Cardiology and the 2019 USA (AHA/ACC/HRS) guidelines recommend the use of AADs for rhythm control in patients with symptomatic AF. This study sought to understand AAD treatment practices and adherence to guidelines across the EU and the USA. Method An online physician survey of cardiologists, cardiac electrophysiologists and interventional electrophysiologists (N = 569) was conducted in the USA, Germany, Italy and the UK. All respondents were actively treating ≥10 AF patients who received drug therapy and/or who had received or were referred for ablation. This extensively detailed survey explored questions on physician demographics, AF types, and drug treatment and ablation practices. Results: Of the responses obtained (1) Amiodarone was used frequently across co-morbidity categories (highest use in those with heart failure with reduced left ventricular ejection fraction [LVEF] [80%]), including in those in which it is not indicated for initial therapy (minimal or no structural heart disease: 26%). Other deviations from guideline recommendations, include: class 1C drugs were used with structural heart disease, including coronary artery disease (CAD) (average class 1C use in CAD-related comorbidities: 6%); sotalol was used with renal dysfunction (22%); and drugs such as sotalol and dofetilide were initiated out of hospital (56% and 17% of respondents, respectively). (2) Nonetheless, a majority of respondents (53%) considered guidelines as the most important non-patient factor in influencing their choice of AF management. (3) Rhythm control was selected more frequently as primary therapy for paroxysmal AF (PAF) (59% of patients) while rate control was used more often for persistent AF (53%). (4) For PAF, AADs were preferred as 1st line more often than ablation, especially if PAF was infrequent and mildly symptomatic (59% of respondents) while ablation was preferred more if frequent symptomatic PAF and for recurrent persistent AF. (5) Rhythm control (AAD or ablation) was chosen in notable numbers for asymptomatic AF and subclinical AF (AADs: 36% and 37%, respectively; ablation: 9% and 14%, respectively). (6) AAD use for those with a first or recurrent episodes of symptomatic AF was 60% or 47%, respectively. (7) Efficacy and safety were chosen as the most important considerations for choice of specific rhythm control therapy (49% and 33%, respectively), and reduction of mortality and cardiovascular hospitalisation (23%) were as important as maintaining sinus rhythm (26%) for rhythm therapy goals. Conclusions Although surveyed clinicians consider guidelines important, deviations in patient types and treatments chosen that compromise safety or were not indicated were common. Findings suggest a lack of understanding of the pharmacology and safe use of AADs, highlighting an important need for further education. Abstract Figure.

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