scholarly journals Reconstitution in Proteoliposomes of the Recombinant Human Riboflavin Transporter 2 (SLC52A2) Overexpressed in E. coli

2019 ◽  
Vol 20 (18) ◽  
pp. 4416 ◽  
Author(s):  
Lara Console ◽  
Maria Tolomeo ◽  
Matilde Colella ◽  
Maria Barile ◽  
Cesare Indiveri
Keyword(s):  
Cardiovascular Disease ◽  
Amino Acids ◽  
Risk Factor ◽  
Biologically Active ◽  
Suitable Model ◽  
Native Protein ◽  
E Coli ◽  
Few Data ◽  
Riboflavin Transport

Background: the SLC52A2 gene encodes for the riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed. It mediates the transport of Riboflavin across cell membranes. Riboflavin plays a crucial role in cells since its biologically active forms, FMN and FAD, are essential for the metabolism of carbohydrates, amino acids, and lipids. Mutation of the Riboflavin transporters is a risk factor for anemia, cancer, cardiovascular disease, neurodegeneration. Inborn mutations of SLC52A2 are associated with Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset. In spite of the important metabolic and physio/pathological role of this transporter few data are available on its function and regulation. Methods: the human recombinant RFVT2 has been overexpressed in E. coli, purified and reconstituted into proteoliposomes in order to characterize its activity following the [3H]Riboflavin transport. Results: the recombinant hRFVT2 showed a Km of 0.26 ± 0.07 µM and was inhibited by lumiflavin, FMN and Mg2+. The Riboflavin uptake was also regulated by Ca2+. The native protein extracted from fibroblast and reconstituted in proteoliposomes also showed inhibition by FMN and lumiflavin. Conclusions: proteoliposomes represent a suitable model to assay the RFVT2 function. It will be useful for screening the mutation of RFVT2.

scholarly journals Adiponectin: Structure, Physiological Functions, Role in Diseases, and Effects of Nutrition

Nutrients ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 1180
Author(s):  
Kayvan Khoramipour ◽  
Karim Chamari ◽  
Amirhosein Ahmadi Hekmatikar ◽  
Amirhosein Ziyaiyan ◽  
Shima Taherkhani ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Amino Acids ◽  
Molecular Weight ◽  
Energy Regulation ◽  
Treatment Interventions ◽  
Adipose Tissues ◽  
Physiological Functions ◽  
Cellular Events ◽  
Healthy Nutrition

Adiponectin (a protein consisting of 244 amino acids and characterized by a molecular weight of 28 kDa) is a cytokine that is secreted from adipose tissues (adipokine). Available evidence suggests that adiponectin is involved in a variety of physiological functions, molecular and cellular events, including lipid metabolism, energy regulation, immune response and inflammation, and insulin sensitivity. It has a protective effect on neurons and neural stem cells. Adiponectin levels have been reported to be negatively correlated with cancer, cardiovascular disease, and diabetes, and shown to be affected (i.e., significantly increased) by proper healthy nutrition. The present review comprehensively overviews the role of adiponectin in a range of diseases, showing that it can be used as a biomarker for diagnosing these disorders as well as a target for monitoring the effectiveness of preventive and treatment interventions.

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 Plasma Branched-Chain Amino Acids and Incident Cardiovascular Disease in the PREDIMED Trial

2016 ◽  
Vol 62 (4) ◽  
pp. 582-592 ◽  
Author(s):  
Miguel Ruiz-Canela ◽  
Estefania Toledo ◽  
Clary B Clish ◽  
Adela Hruby ◽  
Liming Liang ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Amino Acids ◽  
Cardiovascular Death ◽  
Branched Chain Amino Acids ◽  
Control Group ◽  
Cvd Risk ◽  
Hazard Ratios ◽  
Increased Risk ◽  
Branched Chain

Abstract BACKGROUND The role of branched-chain amino acids (BCAAs) in cardiovascular disease (CVD) remains poorly understood. We hypothesized that baseline BCAA concentrations predict future risk of CVD and that a Mediterranean diet (MedDiet) intervention may counteract this effect. METHODS We developed a case-cohort study within the Prevención con Dieta Mediterránea (PREDIMED), with 226 incident CVD cases and 744 noncases. We used LC-MS/MS to measure plasma BCAAs (leucine, isoleucine, and valine), both at baseline and after 1 year of follow-up. The primary outcome was a composite of incident stroke, myocardial infarction, or cardiovascular death. RESULTS After adjustment for potential confounders, baseline leucine and isoleucine concentrations were associated with higher CVD risk: the hazard ratios (HRs) for the highest vs lowest quartile were 1.70 (95% CI, 1.05–2.76) and 2.09 (1.27–3.44), respectively. Stronger associations were found for stroke. For both CVD and stroke, we found higher HRs across successive quartiles of BCAAs in the control group than in the MedDiet groups. With stroke as the outcome, a significant interaction (P = 0.009) between baseline BCAA score and intervention with MedDiet was observed. No significant effect of the intervention on 1-year changes in BCAAs or any association between 1-year changes in BCAAs and CVD were observed. CONCLUSIONS Higher concentrations of baseline BCAAs were associated with increased risk of CVD, especially stroke, in a high cardiovascular risk population. A Mediterranean-style diet had a negligible effect on 1-year changes in BCAAs, but it may counteract the harmful effects of BCAAs on stroke.

scholarly journals Anxiety as a risk factor for cardiovascular disease independent of depression: A narrative review of current status and conflicting findings

2021 ◽  
Vol 8 (1) ◽  
pp. 205510292098746
Author(s):  
Håvard R Karlsen ◽  
Florian Matejschek ◽  
Ingvild Saksvik-Lehouillier ◽  
Eva Langvik
Keyword(s):  
Cardiovascular Disease ◽  
Risk Factor ◽  
Study Design ◽  
Empirical Support ◽  
Narrative Review ◽  
Current Status ◽  
Depression And Anxiety ◽  
Evidential Support ◽  
Meta Analyses

The aim of this paper is to summarise and evaluate the empirical support for the association between anxiety and cardiovascular disease (CVD) and to address challenges related to method and study design. We review results from meta-analyses and more recent findings on the association of anxiety and the risk of CVD. Depression and anxiety are often listed as psychosocial risk markers of CVD, but the role of anxiety as a risk factor for CVD has not received the same evidential support as the effects of depression. Through a narrative review we identified six meta-analyses as well as 15 recent large studies of anxiety and CVD that we summarise. Some of the conflicting findings may be artefacts of study design or population the sample is drawn from. Researchers should take care to be population specific, measurement specific and outcome specific, and to control for comorbid depression.

scholarly journals Characterization of Soluble, Disulfide Bond-Stabilized, Prokaryotically Expressed Human Thyrotropin Receptor Ectodomain*

Endocrinology ◽  
1997 ◽  
Vol 138 (2) ◽  
pp. 588-593 ◽  
Author(s):  
Y. Bobovnikova ◽  
P. N. Graves ◽  
H. Vlase ◽  
T. F. Davies
Keyword(s):  
Amino Acids ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Thioredoxin Reductase ◽  
Biologically Active ◽  
Receptor Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fusion Partner ◽  
E Coli ◽  
Recombinant Receptor

Abstract To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein, it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) products. To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21–415) was inserted into the vector pGEX-2TK by directional cloning and used to transform the thioredoxin reductase mutant strain of E. coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm. After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR amino acids 21–35. This showed that over 50% of the expressed hTSHR-ecd was soluble in contrast to expression in a wild-type E. coli (strain αF′), where the majority of the recombinant receptor was insoluble. The soluble recombinant receptor was affinity purified and characterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein. This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes. Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant.

scholarly journals Obesity and Cancer: 27-Hydroxycholesterol, the Missing Link

2020 ◽  
Vol 21 (14) ◽  
pp. 4822
Author(s):  
Arvand Asghari ◽  
Michihisa Umetani
Keyword(s):  
United States ◽  
Cardiovascular Disease ◽  
Estrogen Receptor ◽  
Risk Factor ◽  
Cause Of Death ◽  
The United States ◽  
Missing Link ◽  
Receptor Modulator ◽  
Obesity And Cancer

Obesity is currently affecting more than 40% of the Americans, and if it progresses with this rate, soon one out of two Americans will be obese. Obesity is an important risk factor for several disorders including cardiovascular disease, the first cause of death in the United States. Cancer follows as the second deadliest disease, and a link between obesity and cancer has been suggested. However, it is very hard to establish an exact connection between obesity and cancers due to the multifactorial nature of obesity. Hypercholesterolemia is a comorbidity of obesity and also linked to several cancers. Recently a cholesterol metabolite 27-hydroxycholesterol (27HC) was found to be an endogenous selective estrogen receptor modulator (SERM), which opened new doors toward several interesting studies on the role of this molecule in biological disorders. It is speculated that 27HC might be the missing link in the obesity and cancer chain. Here, we explored the effects of 27-hydroxycholesterol on obesity and cancers with a focus on the SERM capacity of 27HC.

Role of rat intestinal brush-border membrane angiotensin-converting enzyme in dietary protein digestion

1987 ◽  
Vol 253 (6) ◽  
pp. G781-G786 ◽  
Author(s):  
M. Yoshioka ◽  
R. H. Erickson ◽  
J. F. Woodley ◽  
R. Gulli ◽  
D. Guan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Angiotensin Converting Enzyme ◽  
Brush Border ◽  
Dietary Protein ◽  
Brush Border Membrane ◽  
Biologically Active ◽  
Converting Enzyme ◽  
Intestinal Brush Border Membrane ◽  
Intestinal Brush Border

The role of rat intestinal angiotensin-converting enzyme (ACE; E.C 3.4.15.1) in the digestion and absorption of dietary protein was investigated. Enzyme activity was associated with the brush-border membrane fraction, with the highest activity in the proximal to midregion of the small intestine. Preliminary enzyme characterization studies were carried out using purified brush-border membrane preparations. When a variety of N-blocked synthetic peptides were used as potential substrates for ACE, activity was highest with those containing proline at the carboxy terminal position. The hydrolytic rates observed with these prolyl peptides were comparable to those observed when major digestive peptidases of the brush-border membrane such as aminopeptidase N and dipeptidyl aminopeptidase IV were assayed. When isolated rat jejunum was perfused in vivo with solutions of Bz-Gly-Ala-Pro, the dipeptide Ala-Pro was the main hydrolytic product detected in the perfusates. Absorption rates of the constituent amino acids, alanine and proline, depended on the concentration of peptide perfused. Captopril, an active site specific ACE inhibitor, significantly inhibited hydrolysis and absorption of constituent amino acids from Bz-Gly-Ala-Pro. These results show that intestinal brush-border membrane ACE functions as a digestive peptidase in addition to its role as a regulator of biologically active peptides in other tissues.

scholarly journals Role of Burkholderia pseudomallei Sigma N2 in Amino Acids Utilization and in Regulation of Catalase E Expression at the Transcriptional Level

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Duong Thi Hong Diep ◽  
Nguyen Thi Thanh Phuong ◽  
Mya Myintzu Hlaing ◽  
Potjanee Srimanote ◽  
Sumalee Tungpradabkul
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Burkholderia Pseudomallei ◽  
Transcriptional Level ◽  
Chromosome 2 ◽  
E Coli ◽  
Alternative Sigma Factors ◽  
Amino Acid Utilization ◽  
Genes Expression

Burkholderia pseudomallei is the causative agent of melioidosis. The complete genome sequences of this pathogen have been revealed, which explain some pathogenic mechanisms. In various hostile conditions, for example, during nitrogen and amino acid starvation, bacteria can utilize alternative sigma factors such as RpoS and RpoN to modulate genes expression for their adaptation and survival. In this study, we demonstrate that mutagenesis of rpoN2, which lies on chromosome 2 of B. pseudomallei and encodes a homologue of the sigma factor RpoN, did not alter nitrogen and amino acid utilization of the bacterium. However, introduction of B. pseudomallei rpoN2 into E. coli strain deficient for rpoN restored the ability to utilize amino acids. Moreover, comparative partial proteomic analysis of the B. pseudomallei wild type and its rpoN2 isogenic mutant was performed to elucidate its amino acids utilization property which was comparable to its function found in the complementation assay. By contrast, the rpoN2 mutant exhibited decreased katE expression at the transcriptional and translational levels. Our finding indicates that B. pseudomallei RpoN2 is involved in a specific function in the regulation of catalase E expression.

Role of endothelin in mediating postmenopausal hypertension in a rat model

2005 ◽  
Vol 288 (1) ◽  
pp. R229-R233 ◽  
Author(s):  
Licy L. Yanes ◽  
Damian G. Romero ◽  
Valeria E. Cucchiarelli ◽  
Lourdes A. Fortepiani ◽  
Celso E. Gomez-Sanchez ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Cardiovascular Disease ◽  
Risk Factor ◽  
Spontaneously Hypertensive Rats ◽  
Renal Cortex ◽  
Hypertensive Rats ◽  
Spontaneously Hypertensive ◽  
Peptide Expression ◽  
Premenopausal Female

Cardiovascular disease is the leading cause of death in women after menopause. Hypertension, a major cardiovascular risk factor, becomes more prevalent after menopause. The mechanisms responsible for the increase in blood pressure (BP) in postmenopausal women are unknown. We have recently characterized the aged, postestrous-cycling (PMR) spontaneously hypertensive rats (SHR) as a model of postmenopausal hypertension. The purpose of the present study was to determine whether endothelin plays a role in the increased BP in PMR. Premenopausal female SHR, aged 4–5 mo (YF), and PMR, aged 16 mo, were studied. Expression of preproendothelin-1 mRNA was not different in either renal cortex or medulla between PMR and YF ( n = 7–8/group). In contrast, ET-1 peptide expression was significantly higher in renal cortex of PMR than in renal cortex of YF, but there was no difference in medullary ET-1. Expression of endothelin ETA receptor (ETAR) mRNA was lower in renal cortex and medulla of PMR than of YF. Additional groups of rats ( n = 6–7/group) were treated for 3 wk with the ETAR antagonist ABT-627 (5 mg·kg−1·day−1). BP was significantly higher in PMR than in YF. ETAR antagonist reduced BP in PMR by 20% to the level found in control YF. ETAR antagonist had no effect on BP in YF. These data support the hypothesis that the increase in BP in PMR is mediated in part by endothelin and the ETAR.

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