Presence Of Homocysteine Research Articles

A lateral flow strip for on-site detection of homocysteine based on a truncated aptamer.

An elevated level of homocysteine (Hcy) in serum is closely related to the development of various diseases. Therefore, homocysteine has been widely employed as a biomarker in medical diagnosis and the on-site detection of homocysteine is highly desired. In this study, a truncated highly specific aptamer for homocysteine was screened and used to design a lateral flow strip (LFS) for the detection of homocysteine. The aptamer was derived from a previously reported sequence. Based on the result of molecular docking, the original sequence was subjected to truncation, resulting in a reduction of the length from 66 nt to 55 nt. Based on the truncated aptamer, the LFS was designed for the detection of homocysteine. In the presence of homocysteine, the aptamer selectively binds to it, releasing cDNA from the aptamer/cDNA duplex. This allows cDNA to bind to the capture probe immobilized on the T zone of the strip, resulting in a red signal on the T zone from gold nanoparticles (AuNPs). The strip enables the visual detection of homocysteine in 5 min. Quantitative detection can be facilitated with the aid of ImageJ software. In this mode, the linear detection range for homocysteine is within 5-50 μM, with a detection limit of 4.18 μM. The strip has been effectively utilized for the detection of homocysteine in human serum. Consequently, the combination of the truncated aptamer and the strip offers a method that is sensitive, quick, and economical for the on-site detection of homocysteine.

The Effect of Homocysteine on the Secretion of Il-1β, Il-6, Il-10, Il-12 and RANTES by Peripheral Blood Mononuclear Cells-An In Vitro Study.

The contemporary theory of the inflammatory-immunological pathomechanism of atherosclerosis includes the participation of interleukin-1β (Il), Il-6, Il-10, Il-12, RANTES, and homocysteine in this process. The knowledge on the direct effect of hyperhomocysteinemia on inflammatory-state-related atherosclerosis is rather scarce. Our study is the first to account for the effects of homocysteine on the secretion of Il-10 and RANTES in vitro conditions. For this purpose, human mitogen-stimulated peripheral blood mononuclear cells (PBMNCs) were cultured in vitro and exposed to homocysteine at high concentrations. Subsequently, the concentrations of cytokines were assayed in the cell culture supernatant using flow cytofluorimetry. It has been shown that, in the presence of homocysteine, the secretion of IL-1, IL-6 and RANTES by PBMNCs was increased, whereas IL-10 concentration was significantly lower than that of the supernatant derived from a mitogen-stimulated cell culture without homocysteine. The secretion of Il-12 by PBMNCs exposed exclusively to mitogen, did not differ from homologous cells also treated with homocysteine. Therefore, in our opinion, high-concentration homocysteine affects the progression of atherosclerosis by increasing the secretion of proinflammatory cytokines secreted by PBMNCs, such as Il-1β, Il-6, RANTES, and by attenuating the secretion of Il-10.

A novel near-infrared fluorescent probe for intracellular detection of cysteine.

Cysteine (Cys) takes part in redox balance in cells as an antioxidant, and imbalance of Cys content in the body can cause a variety of diseases. It is very important to develop a new fluorescent chemosensor to specifically detect Cys intracellular. In this work, a novel NIR fluorescent probe was constructed based on 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indol-3-ium iodide and 5-(4-hydroxyphenyl)furan-2-carbaldehyde. The probe could selectively detect Cys in the presence of homocysteine (Hcy), glutathione (GSH), and other interferences. It also had a number of advantages, including nucleolus-targeting ability, long fluorescence emission wavelength (685nm), low detection limit (56nM), and large Stokes shift (172nm). The probe was employed to enable visualization of Cys in HepG2 cells, and due to its good response in viscous environment, the probe could also locate nucleoli intracellular.

Selective homocysteine detection of nitrogen-doped graphene quantum dots: Synergistic effect of surface catalysis and photoluminescence sensing

As a new photoluminescence functional material, graphene quantum dots have aroused widely entertaining. However, the complicated surficial chemistry micro-circumstance of graphene quantum dots hinders the further study of graphene quantum dots. In this paper, we demonstrate that the selectivity and sensitivity photoluminescence (PL) detection of homocysteine based on the synergistic effect of surface catalysis and photoluminescence sensing for nitrogen-doped graphene quantum dots (N-GQDs). Due to the localized superacid microenvironment of N-GQDs, the homocysteine cyclized into homocysteine thiolactone efficiently and selectively. Meanwhile, the aromatic N in N-GQDs adsorbs homocysteine thiolactone selectively. The following static quenching results in the outstanding selectivity and sensitivity PL quenching of N-GQDs with the presence of homocysteine. The detection limit is 5 × 10−11 M, which is much lower than most reported detection methods. We also demonstrate that N-GQDs are able to display a PL response to homocysteine in the living cells.

Nox4 and soluble epoxide hydrolase synergistically mediate homocysteine-induced inflammation in vascular smooth muscle cells

BackgroundHyperhomocysteinemia leads to a vascular smooth muscle cell (VSMC) inflammatory response. Meanwhile, Nox4 dependent reactive oxygen species (ROS) signaling and soluble epoxide hydrolase (sEH)/epoxyeicosatrienoic acids (EETs) are both involved in vascular inflammation. Herein, we hypothesized that Nox4 and soluble epoxide hydrolase cross regulated during homocysteine-induced VSMC inflammation. Methods and resultsIn cultured VSMCs, the expression of the inflammatory factors VCAM1 and ICAM1 was measured by real-time PCR and Western blotting, while supernatant MCP1 was measured by ELISA. Upon VSMC stimulation with 50 μΜ homocysteine, we observed the VCAM1 and ICAM1 mRNA levels were increased by 1.15 and 1.0 folds, respectively. The MCP1 levels in the supernatant of cultured VSMCs treated with 100 μΜ increased to 1.76 folds. As expected, homocysteine induced Nox4 expression and Nox4-dependent ROS generation. The sEH expression was also upregulated in the presence of homocysteine in a dose-dependent manner. Furthermore, we knocked down Nox4 with siRNA. Knockdown of Nox4 decreased ROS generation and homocysteine-induced sEH expression. Overexpression of Nox4 with an adenovirus stimulated sEH expression. Similarly, knockdown or chemical inhibition of sEH blunted the upregulation of Nox4 by homocysteine. In vivo, in homocysteine-fed mice, concomitant upregulation of Nox4 and sEH was associated with increased VCAM1 and ICAM1 expression in the aortic wall. ConclusionsThe inflammatory response induced by homocysteine in VSMCs was accompanied by Nox4 and sEH upregulation. Nox4 and soluble epoxide hydrolase synergistically contribute to homocysteine-induced inflammation.

Competition of Electrocatalytic Activity and Oxidative Ring Cleavage of Unstable Triazolinediones: The Substituent Effect

Electro-oxidation of urazoles has been investigated in the presence of homocysteine (HCys) to provide insight into the competition of oxidative ring cleavage (ORC) of urazoles and electrocatalytic activity of unstable triazolinediones (TADs), the oxidized form of urazoles. The results reveal TADs promote HCys oxidation despite its high instability due to fast chemical kinetics of ORC. The electrocatalytic behavior of 4-phenylurazole in the presence of HCys and the ORC in the absence of HCys have been conducted over the entire range of pHs. The homogeneous oxidation reaction of HCy with TADs proceeds dramatically in the solution with pH value of 7.0 despite intensive instability of TADs in the natural pH. In addition, electrochemistry of 4-phenylurazole (4-PU) has been evaluated in presence of glutathione (GSH). The redox chemistry of 4-PU is not influenced by presence of glutathione. The voltammetric response of urazoles in HCys and GSH mixture can be utilized for the selective electrochemical detection of HCys. Further, turnover frequency (TOF) has been estimated for some urazoles. The catalytic behavior is affected by the presence of functional groups with electron-withdrawing/donating properties. The oxidative potential of urazoles and ring opening kinetic of TADs have been recognized as two main parameters affected on TOF.

Homocysteine-induced proliferation of vascular smooth muscle cells occurs via PTEN hypermethylation and is mitigated by Resveratrol

Vascular smooth muscle cell (VSMC) proliferation is a primary pathological event in the development of atherosclerosis (AS), and the presence of homocysteine (Hcy) acts as an independent risk factor for AS. However, the underlying mechanisms remain to be elucidated. Phosphatase and tensin homologue on chromosome 10 (PTEN), is endogenously expressed in VSMCs and induces multiple signaling networks involved in cell proliferation, survival and inflammation, however, the specific role of PTEN is still unknown. The present study detected the proliferation ratio of VSMCs following treatment with Hcy and Resveratrol (RSV). In the 100µM Hcy group, the proliferation ratio increased, and treatment with RSV decreased the proliferation ratio induced by Hcy. Reverse transcription‑quantitative polymerase chain reaction and western blotting were used to analyze PTEN expression, RSV treatment was associated with decreased PTEN expression levels in VSMCs. PTEN levels were decreased in Hcy treated cells, and the proliferation ratio of VSMCs were increased following treated with Hcy. To study the mechanism of regulation of PTEN by Hcy, the present study detected PTEN methylation levels in VSMCs, and PTEN DNA methylation levels were demonstrated to be increased in the 100µM Hcy group, whereas treatment with RSV decreased the methylation status. DNA methyltransferase 1 is important role in the regulation of PTEN methylation. Overall, Hcy impacts the methylation status of PTEN, which is involved in cell proliferation, and induces the proliferation of VSMCs. This effect is alleviated by treatment with RSV, which exhibits an antagonistic mechanism against Hcy.

Interaction of Citrate-Capped Gold Nanoparticles with the Selected Amino Thiols for Sensing Applications

The amino thiols, methionine, cysteine, and homocysteine are considered to be important biomarkers for various diseases. In the present study, the authors have demonstrated that the interactions of synthesized citrate-capped gold nanoparticles with these amino thiols at different pH values can be used to develop simple colorimetric methods for their detection in aqueous solutions. The aggregation of the gold nanoparticles by interactions with the thiol group was found to depend strongly on the pH of the amino thiols. At pH 10, the detection of cysteine was possible in the presence of homocysteine at low concentration and similarly homocysteine could be detected in the presence of cysteine at low concentration by thiol-induced aggregation of the gold nanoparticles. Cysteine and methionine were specifically detected by monitoring the aggregation of the gold nanoparticles through the UV–Visible spectra at pH 7 and pH 10 respectively. The increase in the mean hydrodynamic diameter of the particles confirmed the thiol-induced aggregation of the particles both at pH 7 and pH 10. The specific pH-based interactions can form a basis to design simple colorimetric sensors for the amino thiols using as-synthesized citrate-capped gold nanoparticles without further functionalization.

Vascular smooth muscle cell differentiation to an osteogenic phenotype involves matrix metalloproteinase-2 modulation by homocysteine.

Arterial calcification is common in vascular diseases and involves conversion of vascular smooth muscle cells (VSMCs) to an osteoblast phenotype. Clinical studies suggest that the development of atherosclerosis can be promoted by homocysteine (HCY), but the mechanisms remain unclear. Here, we determined whether increases in HCY levels lead to an increase in VSMC calcification and differentiation, and examined the role of an extracellular matrix remodeler, matrix metalloproteinase-2 (MMP-2). Rat VSMCs were exposed to calcification medium in the absence or presence of HCY (10, 100 or 200 μmol/L) or an MMP-2 inhibitor (10(-6) or 10(-5) mol/L). MTT assays were performed to determine the cytotoxicity of the MMP-2 inhibitor in calcification medium containing 200 μmol/L HCY. Calcification was assessed by measurements of calcium deposition and alkaline phosphatase (ALP) activity as well as von Kossa staining. Expression of osteocalcin, bone morphogenetic protein (BMP)-2, and osteopontin, and MMP-2 was determined by immunoblotting. Calcification medium induced osteogenic differentiation of VSMCs. HCY promoted calcification, increased osteocalcin and BMP-2 expression, and decreased expression of osteopontin. MMP-2 expression was increased by HCY in a dose-dependent manner in VSMCs exposed to both control and calcification medium. The MMP-2 inhibitor decreased the calcium content and ALP activity, and attenuated the osteoblastic phenotype of VSMCs. Vascular calcification and osteogenic differentiation of VSMCs were positively regulated by HCY through increased/restored MMP-2 expression, increased expression of calcification proteins, and decreased anti-calcification protein levels. In summary, MMP-2 inhibition may be a protective strategy against VSMC calcification.

Selective electrochemical determination of cysteine with a cyclotricatechylene modified carbon electrode.

We report the selective electrochemical detection of cysteine in the presence of homocysteine and glutathione with the use of an electrode modified with cyclotricatechylene (CTC). A carbon electrode was first modified with cyclotriveratrylene (CTV) and then electrochemically converted into CTC. Using cyclic voltammetry, the redox activity of CTC was investigated along with its electrochemical response to cysteine and the closely related compounds, glutathione and homocysteine which are commonly found in biological media alongside cysteine. The selective detection of cysteine was achieved with the use of the electrocatalytic oxidation reaction and exploiting the different rates of reaction of each thiol with the oxidized CTC via variable scan rate studies. The analytical parameters consisting of sensitivity, range of linear detection, and limit of detection were determined for selective cysteine detection in phosphate buffer solution and tissue culture media where the sensitivity of the system is ca. 0.023 μA μM(-1) and ca. 0.031 μA μM(-1) with a limit of detection of ca. 0.6 μM and ca. 0.9 μM for buffer solution and tissue culture media respectively. Practical assessment of this analytical method was carried out in mixed solutions containing a combination of cysteine, homocysteine and glutathione in both media. The determined results agree well with the added cysteine content. This work presents a novel way of utilizing CTC into detecting cysteine, and is well-suited for bio-marker sensing.

The necrotrophic effector protein SnTox3 re-programs metabolism and elicits a strong defence response in susceptible wheat leaves.

BackgroundThe fungus Stagonospora nodorum is a necrotrophic pathogen of wheat. It causes disease by secreting proteinaceous effectors which interact with proteins encoded by dominant susceptibility genes in the host. The outcome of these interactions results in necrosis, allowing the fungus to thrive on dead plant material. The mechanisms of these effectors though are poorly understood. In this study, we undertake a comprehensive transcriptomics, proteomic and metabolomic approach to understand how a susceptible wheat cultivar responds to exposure to the Stagonospora nodorum effector protein SnTox3.ResultsMicroarray and proteomic studies revealed that SnTox3 strongly induced responses consistent with those previously associated with classical host defence pathways including the expression of pathogenicity-related proteins and the induction of cell death. Collapse of the photosynthetic machinery was also apparent at the transcriptional and translational level. SnTox3-infiltrated wheat leaves also showed a strong induction of enzymes involved in primary metabolism consistent with increases in hexoses, amino acids and organic acids as determined by primary metabolite profiling. Methionine and homocysteine metabolism was strongly induced upon exposure to SnTox3. Pathogenicity in the presence of homocysteine was inhibited confirming that the compound has a role in plant defence. Consistent with the strong defence responses observed, secondary metabolite profiling revealed the induction of several compounds associated with plant defence, including the phenylpropanoids chlorogenic acid and feruloylquinic acid, and the cyanogenic glucoside dhurrin. Serotonin did not accumulate subsequent to SnTox3 infiltration.ConclusionsThese data support the theory that the SnTox3 effector protein elicits a host cell death response to facilitate the pathogen’s necrotrophic infection cycle. Our data also demonstrate that the mechanism of SnTox3 appears distinct from the previously characterised Stagonospora nodorum effector SnToxA. Collectively, this comprehensive analysis has advanced our understanding of necrotrophic effector biology and highlighted the complexity of effector-triggered susceptibility.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0215-5) contains supplementary material, which is available to authorized users.

Differential regulation of DNA methylation versus histone acetylation in cardiomyocytes during HHcy in vitro and in vivo: an epigenetic mechanism

The mechanisms of homocysteine-mediated cardiac threats are poorly understood. Homocysteine, being the precursor to S-adenosyl methionine (a methyl donor) through methionine, is indirectly involved in methylation phenomena for DNA, RNA, and protein. We reported previously that cardiac-specific deletion of N-methyl-d-aspartate receptor-1 (NMDAR1) ameliorates homocysteine-posed cardiac threats, and in this study, we aim to explore the role of NMDAR1 in epigenetic mechanisms of heart failure, using cardiomyocytes during hyperhomocysteinemia (HHcy). High homocysteine levels activate NMDAR1, which consequently leads to abnormal DNA methylation vs. histone acetylation through modulation of DNA methyltransferase 1 (DNMT1), HDAC1, miRNAs, and MMP9 in cardiomyocytes. HL-1 cardiomyocytes cultured in Claycomb media were treated with 100 μM homocysteine in a dose-dependent manner. NMDAR1 antagonist (MK801) was added in the absence and presence of homocysteine at 10 μM in a dose-dependent manner. The expression of DNMT1, histone deacetylase 1 (HDAC1), NMDAR1, microRNA (miR)-133a, and miR-499 was assessed by real-time PCR as well as Western blotting. Methylation and acetylation levels were determined by checking 5'-methylcytosine DNA methylation and chromatin immunoprecipitation. Hyperhomocysteinemic mouse models (CBS+/-) were used to confirm the results in vivo. In HHcy, the expression of NMDAR1, DNMT1, and matrix metalloproteinase 9 increased with increase in H3K9 acetylation, while HDAC1, miR-133a, and miR-499 decreased in cardiomyocytes. Similar results were obtained in heart tissue of CBS+/- mouse. High homocysteine levels instigate cardiovascular remodeling through NMDAR1, miR-133a, miR-499, and DNMT1. A decrease in HDAC1 and an increase in H3K9 acetylation and DNA methylation are suggestive of chromatin remodeling in HHcy.

Selective electrochemical determination of homocysteine in the presence of cysteine and glutathione

The electrochemical oxidation of 3,5-di-tert-buthylcatechol in the presence of homocysteine was used for the selective electrochemical determination of homocysteine in the presence of cysteine and glutathione at a glassy carbon and a glassy carbon electrode modified with carbon nanotube. The results revealed that the electrochemically generated 3,5-di-tert-butylcyclohexa-3,5-diene-1,2-dione exhibits high catalytic activity toward homocysteine oxidation at reduced over-potential and low catalytic activity for oxidation of cysteine. The catalytic activity 3,5-di-tert-butylcyclohexa-3,5-diene-1,2-dione toward cysteine was suppressed in the presence of 4-N,N-dimethylaminocinnamaldehyde. Contrary to homocysteine and cysteine, the reaction of glutathione with 3,5-di-tert-butylcyclohexa-3,5-diene-1,2-dione is a substituation reaction. This method exhibits three dynamic linear ranges of 2.5 to 10μmolL−1, 10 to 100μmolL−1 and 100 to 1000μmolL−1, and a lower detection limit (3σ) of 0.89±3.53% μmol L−1 for homocysteine.

In-vitro study of homocysteine and aspirin effects on fibrinolysis

Some studies suggest that increased homocysteine in blood leads to alterations in coagulation and fibrinolysis; however, the precise mechanism is not clear. The aim of this study was to compare different concentrations of homocysteine and aspirin on fibrinolysis in the plasma of healthy individuals in vitro. Different concentrations of homocysteine (200, 100, and 50 μmol/l) and aspirin (100, 10, and 1 mg/l) were added to the healthy people plasma citrate. They were incubated at 37°C for 24 h. Then, fibrinolysis parameters were analyzed by the turbidimetric procedure at 405 nm. The independent-samples t-test was utilized to compare them (P < 0.05). Findings revealed that homocysteine at 200 μmol/l with aspirin 100 ml/g had significant changes in the lysis maximum velocity (0.150 ± 0.002), half-lysis time (218 ± 5.77), the total lysis time (446 ± 5.77), and lag time in lysis (119 ± 3.60), compared to homocysteine at 200 μmol/l lysis maximum velocity (0.110 ± 0.002), half-lysis time (278 ± 7.63), the total lysis time (515 ± 14.29), and lag time in lysis (176 ± 3.60), respectively (P < 0.05). Homocysteine at 200 μmol/l with aspirin 1 ml/g did not significantly change in either parameter (P > 0.05). Homocysteine at 50 μmol/l with aspirin (100, 10, and 1 mg/l) had significant changes in all fibrinolysis parameters (P < 0.05), compared to homocysteine at 50 μmol/l. The other concentrations were compared in the same way. Aspirin (more than 1 mg/l) had more effect on higher concentrations of homocysteine. Aspirin increased velocity of clot lysis and decreased lysis time of clot in the presence of homocysteine.

Homocysteine-functionalized silver nanoparticles for selective sensing of Cu2+ ions and Lidocaine hydrochloride

A sensitive and selective colorimetric detection method for Cu2+ ions and lidocaine hydrochloride (LC-HCl) has been developed in this study. Herein, homocysteine-functionalized silver nanoparticles (Hcys-AgNPs), prepared by reducing AgNO3 with sodium borohydride in the presence of homocysteine, played an important role in this strategy. Aggregation of Hcys-AgNPs was induced in the presence of Cu2+ ions, yielding a dramatic color change from deep brown to bright yellow. In contrast, once both Cu2+ ions and LC-HCl existed together, the Hcys-AgNPs showed little aggregation, leading to much less color change compared with that in the presence of Cu2+ ions alone. This investigation demonstrated that plasmon resonance scattering (PRS) signals in the corresponding plasmon resonance absorption (PRA) region could be monitored using a common spectrofluorometer, and be enhanced by the Cu2+-induced aggregation of Hcys-AgNPs. Significantly, the PRS intensity characterized at 571.0nm was found to be proportional to the concentration of Cu2+ ions in the range of 30nM–1μM with the limit of determination of 3.2nM (3σ) and the concentration of LC-HCl in the range of 50–500nM with the limit of determination of 4.6nM (3σ) under optimal conditions. Additionally, the Hcys-AgNPs bound by Cu2+ represented excellent selectivity compared to other metal ions (Fe2+, Mg2+, Mn2+, Hg2+, Zn2+, Ni2+, Co2+, Pb2+, Ba2+, Ca2+, Cd2+, Al3+, Fe3+, Cr3+ and K+), and Procaine hydrochloride (PC-HCl) indicated no interference to LC-HCl. Finally, the method was applied to detect Cu2+ ions of water samples and LC-HCl in rat serum and human urine with low interference compared with HPLC, suggesting we provided a simple and reliable method towards detecting both Cu2+ ions and LC-HCl.

Effect of bisphosphonates on gene expression of fibroblasts cultured in the presence of homocysteine

Effect of bisphosphonates on gene expression of fibroblasts cultured in the presence of homocysteine

Feasibility of Raman spectroscopic analysis of skin to discriminate between healthy and diseased organic matrix: A case study

Feasibility of Raman spectroscopic analysis of skin to discriminate between healthy and diseased organic matrix: A case study

Mechanical properties of articular cartilage/bone interface assessed by scanning acoustic microscopy

Mechanical properties of articular cartilage/bone interface assessed by scanning acoustic microscopy

Estrogen signaling to bone is not amplitude‐modulated in adult rats

Estrogen signaling to bone is not amplitude‐modulated in adult rats

Colorimetric Assay for S-Adenosylhomocysteine Hydrolase Activity and Inhibition Using Fluorosurfactant-Capped Gold Nanoparticles

This study reports a simple colorimetric method for the sensitive detection of S-adenosylhomocysteine hydrolase (SAHH) activity and inhibition using fluorosurfactant-capped gold nanoparticles (FSN-AuNPs). FSN stabilizes the AuNPs against conditions of high ionic strength, and FSN-AuNPs are merely aggregated in the presence of homocysteine (HCys) and cysteine. Because of this feature, FSN-AuNPs were found to be dispersed in the presence of S-adenosylhomocysteine (SAH) that lacks a free thiol group. After SAHH catalyzed the hydrolysis of SAH, the produced HCys molecules were bound to the surface of AuNPs through the formation of Au-S bonds. As a result, the nanoparticle (NP) aggregation occurred through electrostatic attraction between each HCys-attached AuNP. This approach had a minimum detectable concentration of 100 units/L (~6 nM). Additionally, because adenosine analogs are capable of inhibiting SAHH activity, the addition of adenosine analogs to a solution containing SAH and SAHH resulted in the suppression of hydrolyzed SAH-induced NP aggregation. Adenosine analogs exhibited the following trend in the half-maximal inhibitory concentrations: adenosine > adenosine monophosphate > adenosine diphosphate ~ adenosine triphosphate. We have demonstrated that the combination of SAHH inhibition and FSN-AuNPs can be utilized for the selective detection of adenosine.