Exogenous Cys Research Articles

Exogenous cysteine alleviates mercury stress by promoting antioxidant defence in maize (Zea mays L.) seedlings

Mercury (Hg) is one of the most important environmental pollutants that negatively affects plant growth and development. Cysteine (Cys) plays an important role in plant response to various environmental stress factors. In the present study, the alleviation of Hg stress through exogenous Cys treatment to maize seedlings was evaluated. For this purpose, a hydroponic experiment was set up to investigate the effect of HgCI2 (100 μM) and in combination with Cys (200 μM) on plant growth, total chlorophyll content, reactive oxygen species, antioxidant enzyme activities, and mRNA expression levels of some antioxidant genes in maize seedlings. The results showed that HgCI2 treatment significantly decreased both root and shoot growth and total chlorophyll content, also increased the malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide levels (O2.–) in maize seedlings. After treatment with 200 μM exogenous Cys combined with 100 μM HgCl2, root and shoot growth and total chlorophyll content increased and the concentration of MDA, H2O2, O2 .– in seedlings notably decreased and catalase (CAT), glutathione reductase (GR), superoxide dismutase (SOD), and peroxidase (POD) activities in seedlings increased significantly. In addition, HgCI2 treatment alone or combination with Cys markedly increased the root and leaf Hg content. The higher amounts of Hg accumulated in the roots of the seedlings. Furthermore, qRT-PCR results showed that the mRNA levels of CAT, GR, and SOD genes were up-regulated at HgCI2 + Cys treatment groups compared to the HgCI2 treatment alone. The results of the study indicated that exogenous Cys improved resistance to Hg-stress in maize seedlings by activating antioxidant defence system.

A curcumin-analogous fluorescent sensor for cysteine detection with a bilateral-response click-like mechanism.

A novel curcumin-analogous fluorescent sensor, DNP, was developed for cysteine detection with a bilateral-response click-like mechanism. DNP indicated high selectivity and practical sensitivity. It could recognize Cys from other biologically relevant molecules, especially, from GSH and Hcy. The most interesting point was that, with typical azide groups for sensing, DNP indicated a covalent binding procedure with Cys instead of a presupposed simple reduction for reductive sulfide. Moreover, the recognition occurred at both sides of the sensor. DNP could be utilized into the detection of endogenous and exogenous Cys in living cells. Though the specific optical performances of DNP still need optimization, this work supplied novel information for broadening the vision on fluorophores and mechanisms, for the monitoring of Cys and even other sulfur-containing species.

Highly selective discrimination of cysteine from glutathione and homo-cysteine with a novel AIE-ESIPT fluorescent probe

Biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play pivotal roles in numerous physiological events, however, due to the similar structures and reactivities, differentiation of these biothiols remains a challenging task. Herein, we reported a maleimide-appended fluorescent probe ABTT-MA capable of specific sensing of Cys over other structure-alike biothiols with an ultralow detection limit (9.4 nM). Via a series of investigations into the mechanism, the high selectivity toward Cys is ascribed to a Michael addition/S,N-intramolecular rearrangement cascade reaction, in which the involvement of the mercapto and the adjacent amino groups is necessary to proceed the sensing process. Given this presupposition, Cys could be kinetically discriminated from Hcy and GSH. Upon the addition of Cys, a strong fluorescence appeared and the intensity at 503 nm was correlated with Cys concentration linearly over a wide range (0–10 μM). Thanks to the aggregation-induced emission (AIE) and excited-state intramolecular proton transfer (ESIPT) activity, the selective detection of Cys was realized not only in aqueous media but in the paper-based point-of-care test (POCT). Significantly, the practicability of ABTT-MA in exogenous and endogenous Cys imaging in living cells was elucidated as well.

A simple highly selective probe for discriminative visualization of endogenous cysteine, homocysteine and glutathione in living cells via three separated fluorescence channels

Biothiols, as basic biological reactive sulfur species, perform vital actions in many critical physiological processes. Simultaneous detection and direct visualization of intracellular biothiols has great significance to figure out their metabolic mechanisms and cellular functions in living organism. However, it is an enormous challenge to selectively sense biothiols, troubled by their similar chemical structures and properties. In this work, we reported a novel chlorinated coumarin based multi-signal fluorescent probe (CC) for discriminative detection of cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) through three different emission channels. CC exhibited favorable sensing properties towards biothiols such as large fluorescence enhancement, low limits of detection (6, 3 and 200 nM for Cys, Hcy and GSH), fast response speed (15 min) and pretty good water solubility. Moreover, it was also utilized for discriminative visualization of endogenous and exogenous Cys, Hcy and GSH in A375 cells and Tcam-2 cells successfully.

“Turn-on” fluorescence probe for selective recognition of endogenous and exogenous cysteine in cells

Cysteine (Cys) plays an important role in many physiological and pathological processes, and is closely related to many diseases. Therefore, it is necessary to develop fluorescent probes that specifically monitor Cys in vivo, which also helps to further understand the pathological process of related diseases. However, the fluorescent probes reported so far rarely distinguish Cys from the other two biothiols. Therefore, a fluorescent probe with high selectivity and excellent cell membrane permeability, (E)-3-(6-bromopyridin-2-yl)-1-(pyren-3-yl)prop-2-en-1-one (Probe), has been designed for monitoring endogenous and exogenous Cys in HeLa cells.

Ultrasound-promoted specific chiroptical sensing of cysteine in aqueous solution and cells

The quantity and chirality of cysteine (Cys) are correlated with diseases and have crucial implications in physiology and pathology. A selective chiral sensor for Cys based on electronic circular dichroism and UV detection was developed, and the sensing reaction takes place rapidly in aqueous solution with the aid of ultrasound. This sensing approach can simultaneously determine the absolute optical purity and total concentration of Cys. The limit of detection is 0.25 μM, and the detection is not impeded by other naturally occurring amino acids or common biothiols. This method is simple to operate and has the advantages of fast detection, high sensitivity and selectivity, and the measurement error is no more than 4%, with application potential for high-throughput screening. A kit containing this sensor was prepared and successfully applied in a sensing study of cells and peptides containing N-terminal Cys residues to detect endogenous and exogenous Cys in cells and determine the absolute configuration of N-terminal Cys in polypeptides. This work has important application prospects for the study of Cys with the aim of diagnosing related diseases.

1,8-Naphthalimide-based dual-response fluorescent probe for highly discriminating detection of cys and H2S

Hydrogen sulfide (H2S) and cysteine (Cys) are ubiquitous biological thiols in physiological and pathological processes of living systems. Their aberrant concentration levels are associated with many diseases. Hence, we developed a water-soluble fluorescent (FL) probe for H2S and Cys response. The probe was synthesized by two steps of reaction coupling between a triethylene glycol naphthalimide dye and 7-chlorobenzofurazan-4-sulfonyl chloride (CBD) dye linked by a rigid piperazine group. The probe displayed dual fluorescence emission for Cys (λem = 571 nm) and H2S (λem = 535 nm) detection, accompanied by different absorbance changes for Cys (λex = 405 nm) and H2S (λex = 486 nm). The probe could distinguish Cys and H2S from other biological thiols (GSH, Hcy) with longer wavelength or stronger fluorescence emission. Moreover, the probe was allowed to sense the exogenous or endogenous H2S and Cys with dual-emission signal (blue and red channel) in living MCF-7 cells.

Highly specific monitoring and imaging of endogenous and exogenous cysteine in living cells

Cys is one of the important biothiols and its abnormal concentration may pose a threat to human health. Therefore, the monitoring of Cys in organisms is of great significance. GSH and Hcy, as the other two biothiols, have similar chemical structures and active sites to Cys. Consequently, developing fluorescent probes to independently detect Cys has become a challenging problem. Keeping this in mind, α-β unsaturated ketone as a recognition group was integrated into the coumarin group skeleton to synthesize a fluorescent probe SC. After the nucleophilic addition reaction of Cys with SC, the conjugated system of SC was blocked and the fluorescent enhanced obviously. SC was able to detect Cys specifically under the same excitation with a low detection limit (11.1 nM). SC showed a rapid respond to Cys (120 s) and good fluorescent stability over a wide pH range. In addition, it achieved extracorporeal circulation in the presence of H2O2 or NEM. In the end, SC could be applied to detecting endogenous and exogenous Cys under biological condition due to its slight cytotoxicity and good biocompatibility. This provided a powerful tool for studying the physiological function of Cys exclusively.

A novel probe for colorimetric and near-infrared fluorescence detection of cysteine in aqueous solution, cells and zebrafish.

Cysteine(Cys) is tightly related to physiological and pathological of human, and the imbalance of concentration of cysteine in the intracellular are associated with many diseases. Here, a novel NIR fluorescent probe TCF-Cys was designed and synthesized, and both the optimal excitation and emission wavelength of them were between 650 and 900 nm, that within the "optical window" of biological tissues. In aqueous solution, TCF-Cys, which with an acrylate extremity as a recognizing unit, exhibited excellent "turn-on" fluorescence response for Cys superior to other amino acids and thiols with a limit of detection of 0.1323 μM. Moreover, as an excellent naked-eye colorimetric indicator, TCF-Cys could effectively distinguishing the Cys, Hcy and GSH in aqueous solution through color change. Then, the response mechanism of TCF-Cys for Cys was revealed by TLC, 1H NMR, HPLC, HRMS and DFT calculation. Finally, TCF-Cys was successfully employed to fluorescence specifically map of exogenous and endogenous Cys in living cells and zebrafish with low toxicity.

Rational design of cysteine-specific ratiometric probe based on steric hindrance effect and its biological application

Based on our previous work, we modified the coumarin derivate with orthotopic pinacol which can promote the specificity of the probe toward Cysteine (Cys) via steric-hinerance effect to develop a new Cys specific ratiometric fluorescent probe with α, β-unsaturated ketone as the reaction site. The nucleophilic addition reaction between Cys and α, β-unsaturated ketone rearranged the conjugation system of the probe which induced the hypochromatic shift of the fluorescent emission. The probe features good water solubility and the detection process reaches equilibrium within 10 min. Notably, the fast response would provide better precision because of the oxidizable nature of Cys. The fluorescence ratio of the probe increased 9-fold after the reaction and the corresponding detection limit was calculated to be 14 μM. Furthermore, bioimaging experiment results indicated that the probe could detect exogenous and endogenous Cys specifically through the ratiometric manner.

Dual-Site and Dual-Excitation Fluorescent Probe That Can Be Tuned for Discriminative Detection of Cysteine, Homocystein, and Thiophenols.

Thiols play a vital role in both the physiological process and organic synthesis field, including aliphatic thiols (e.g., Cys, Hcy, and GSH) and thiophenols. As a result of the similarities of thiols in terms of molecular structure and chemical properties, it is difficult for conventional fluorescent probes to distinguish them, which hinders the progress of biological and pathological research. Keeping this in mind, a dual-site and dual-excitation fluorescent probe (YY) was designed to distinguish among Cys, Hcy, and thiophenols by three different reaction paths. When excited at 470 nm, YY only exhibits a fluorescence OFF-ON response toward thiophenols. However, when excited at 453 nm, YY not only displays a fluorescence OFF-ON response toward Hcy and thiophenols (λem = 499 and 561 nm) but also presents a two-stage fluorescence response toward Cys, which possesses a fluorescence OFF-ON response in the first stage (λem = 501 nm) and then a fluorescence ON-OFF response in the second stage (λem = 556 nm). This specific fluorescence response indicates that YY has ability to overcome the above-mentioned challenge to achieve discriminative detection of Cys, Hcy, and thiophenols qualitatively, which promotes the study of thiols in the fields of physiology and pathology. Furthermore, cell-imaging studies show that YY can be applied to the imaging of exogenous Cys, Hcy, and thiophenols through two different emission channels.

A simple but effective fluorescent probe with large stokes shift for specific detection of cysteine in living cells

A novel 4′-hydroxybiphenyl-4-carbonitrile-based fluorescent probe, 1, for selective detection of cystein (Cys) over homocystein (Hcy) and glutathione (GSH) was developed. This probe had simple structure and could be easily synthesized with good yield from commercially available materials. Moreover, probe 1 showed a remarkable large Stokes shift (180 nm) and displayed a rapid (5 min) and highly sensitive response (the detection limit was 0.15 μM) for Cys with fluorescence turn-on signal changes (142-fold fluorescence enhancement). Importantly, probe 1 could be used to detect and image both exogenous and endogenous Cys in living A549 cells.

A Multi-signal Fluorescent Probe with Multiple Binding Sites for Simultaneous Sensing of Cysteine, Homocysteine, and Glutathione.

A novel fluorescent probe was developed by integrating chlorinated coumarin and benzothiazolylacetonitrile and exploited for simultaneous detection of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Featuring four binding sites and different reaction mechanisms for different biothiols, this probe exhibited rapid fluorescence turn-on for distinguishing Cys, Hcy, and GSH with 108-, 128-, 30-fold fluorescence increases at 457, 559, 529 nm, respectively, across different excitation wavelengths. Furthermore, the probe was successfully applied to the fluorescence imaging of endogenous Cys and GSH and exogenous Cys, Hcy, and GSH in living cells.

A Multi‐signal Fluorescent Probe with Multiple Binding Sites for Simultaneous Sensing of Cysteine, Homocysteine, and Glutathione

AbstractA novel fluorescent probe was developed by integrating chlorinated coumarin and benzothiazolylacetonitrile and exploited for simultaneous detection of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Featuring four binding sites and different reaction mechanisms for different biothiols, this probe exhibited rapid fluorescence turn‐on for distinguishing Cys, Hcy, and GSH with 108‐, 128‐, 30‐fold fluorescence increases at 457, 559, 529 nm, respectively, across different excitation wavelengths. Furthermore, the probe was successfully applied to the fluorescence imaging of endogenous Cys and GSH and exogenous Cys, Hcy, and GSH in living cells.

A near-infrared fluorescent probe based on BODIPY derivative with high quantum yield for selective detection of exogenous and endogenous cysteine in biological samples

Cysteine (Cys) is involved in cellular growth and Cys deficiency is related with many diseases. So far, a number of fluorescent probes have been constructed for the detection of Cys successfully. However, the probes are difficult to discriminate Cys from Hcy and the emission wavelength of the probes is in ultraviolet or visible range. Herein, a NIR fluorescent probe named NIR-BODIPY-Ac is synthesized and used to detect Cys. The emission wavelength of the probe is at 708 nm that belongs to near-infrared (NIR) region by attaching indolium to BODIPY core, which is suitable for bioimaging in vivo. Moreover, the probe exhibits high fluorescence quantum yield (Φ = 0.51) after the addition of Cys and high sensitivity toward Cys with 81-fold fluorescence enhancement. The linear range of the probe for Cys covers from 0.2 to 30 μM with a detection limit of 0.05 μM. Furthermore, the probe shows high selectivity towards Cys owing to the fact that there is more fast reaction rate between the probe and Cys than that of Hcy. In particular, the NIR fluorescent probe is applied for the detection of exogenous and endogenous Cys in biological samples such as cell, tissue and mouse with satisfactory results.

Benzothiazole‐Pyimidine‐Based BF2 Complex for Selective Detection of Cysteine

Due to the similar structure and reactivity of cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), the simultaneous discrimination of Cys over Hcy and GSH by a single fluorescent sensor is still a great challenge. In this work, a benzothiazole-pyimidine-based boron difluoride complex (BPB) was developed as a new fluorescent sensor for Cys. The sensor exhibits a highly selective "turn-on" response to cysteine over Hcy, GSH and other amino acids in aqueous solution at physiological pH. The observed pseudo-first-order rate constant for the reaction of BPB with Cys was calculated to be about 0.062 min(-1) . The detection limit of this sensor for Cys was determined to be 332 nm, and bioimaging of exogenous Cys by this sensor was successfully applied in living cells, thus indicating that this sensor holds great potential for biological applications.

PLASMA SULFURED AMINO ACID RESPONSES TO METHIONINE‐LOADING TEST IN HIV+ PATIENTS

HIV+ patients present low plasma levels of GSH which could be normalized through dietary supplements with either N‐acetylcysteine (NAC) or glutamine (Gln). Nevertheless, normal Cys levels were not obtained in such situations and lower trans‐sulfuration pathway activity could be the cause. It has been referred that after a methionine load (MetLo) mostly of Cys comes from trans‐sulfuration pathway. The study aimed to investigate the responses of Cys to the MetLo in healthy control (Co) and HIV+ dietary‐supplemented (NAC) subjects. The MetLo (0.1g Met/kg mass weight) was given after overnight fasting to 20 non‐ HIV+ Co and 12 HIV+ HAART‐treated patients. Blood samples were taken before, 2h and 4h after MetLo in different 7‐d dietary situations, with NAC (1g/d) or with their usual diet (DO). The amino acids (Met, Hcy, Cys and Tau) and GSH were determined by HPLC and their inflow rate into circulation (plasma) estimated by the area under the curve (AUC). The difference between groups and diets were compared (p<0.05). When compared to DO the additional effects of MetLo to the NAC supplementation were markedly noted in HIV+ by increasing AUC of Hcy, Cys, Tau and GSH. Met matched the Co and Cys was found lower in both situations while GSH surpassed the Co values. When associated with NAC supplementation the MetLo increased Met probably by the Met spared effect of Cys and then increased Hcy probably by reducing remethylation activity. At the same time, trans‐sulfuration decreased probably feedbacking exogenous Cys. Thus, the failure of restoring normal Cys by MetLo in addition to NAC, in HIV+ patients seems related to increased flux of Cys into GSH and Tau pathways to strengthen the cell antioxidant capacity against the HIV progression. (FAPESP, CAPES and CNPq)

Effects of exogenous glutathione and cysteine on growth, lead accumulation, and tolerance of Iris lactea var. chinensis

Effects of exogenous reduced glutathione (GSH) and cysteine (Cys) on growth, lead (Pb) accumulation, and nonprotein thiol (NPT) contents of Iris lactea var. chinensis under 100 and 500 mg L(-1) Pb stress were studied. Our results showed that 500 mg L(-1) Pb stress caused a dramatical decline in fresh weights, while the reduction of aboveground biomass was alleviated by exogenous GSH and Cys even though keeping higher Pb contents in roots and shoots. Exogenous GSH and Cys could enhance Pb accumulation in the shoots and roots compared with single Pb treatment. The promoting effect of GSH to Pb accumulation was larger than the effect of Cys, and the Pb contents in the shoots and roots treated with 500 mg L(-1) Pb + GSH reached 1,712 and 14,603 mg kg(-1), about 4.19 and 2.78 times of single 500 mg L(-1) Pb treatment, respectively. Microscopic imaging of Pb in roots and leaves showed that higher intensive fluorescence was observed in cell wall of root epidermis, stele, vascular tissues of the roots, and sclerenchyma cells of leaves treated with 500 mg L(-1) Pb + GSH and treated with 500 mg L(-1) Pb + Cys. Exogenous GSH had an apparent promoting effect on root and shoot GSH synthesis, while exogenous Cys reduced the synthesis of cellular GSH in shoot and increased Cys contents. Pb only induced the synthesis of phytochelatin (PC)2 in roots, and the PC2 content declined in GSH- and Cys-treated plant roots. These results suggested that GSH synthesis was a more effective approach to improve Pb accumulation and translocation of I. lactea var. chinensis. Further analysis of protein expression in plants by exogenous GSH and buthionine sulfoximine (BSO) application showed that the proteins regulated by GSH and BSO may constitute various enzymes involved in GSH biosynthesis and play certain roles in Pb accumulation and tolerance of I. lactea var. chinensis.

Cardiovascular actions of l-cysteine and l-cysteine sulfinic acid in the nucleus tractus solitarius of the rat

The sulfur-containing excitatory amino acid (EAA) L-cysteine sulfinic acid (CSA), a neurotransmitter candidate, is endogenously synthesized from L-cysteine (Cys). Exogenous Cys administration into the brain produces cardiovascular effects; these effects likely occur via synaptic stimulation of central nervous system (CNS) neurons that regulate peripheral cardiovascular function. However, the cardiovascular responses produced by CNS Cys administration could result from CSA biosynthesized in synapse. The present study examined the role of CSA in Cys-induced cardiovascular responses within the nucleus tractus solitarius (NTS) of anesthetized rats. The NTS receives input from various visceral afferents that gate autonomic reflexes, including cardiovascular reflexes. Within the NTS, both Cys and CSA microinjections produced decrease responses in arterial blood pressure and heart rate that were similar to those produced by L-glutamate. Co-injection of the ionotropic EAA receptor antagonist kynurenic acid abolished Cys-, but not CSA-, induced cardiovascular responses. This finding suggests that only Cys-induced cardiovascular responses are mediated by kynurenate-sensitive receptors. This study provides the first demonstration that Cys- and CSA-induced cardiovascular responses occur via different mechanisms in the NTS of rats. Further, this study also indicates that Cys-induced cardiovascular responses do not occur via CSA. Thus, within the NTS, endogenous Cys and/or CSA might be involved in cardiovascular regulation.

Size and Conformation Limits to Secretion of Disulfide-bonded Loops in Autotransporter Proteins

Autotransporters are a superfamily of virulence factors typified by a channel-forming C terminus that facilitates translocation of the functional N-terminal passenger domain across the outer membrane of Gram-negative bacteria. This final step in the secretion of autotransporters requires a translocation-competent conformation for the passenger domain that differs markedly from the structure of the fully folded secreted protein. The nature of the translocation-competent conformation remains controversial, in particular whether the passenger domain can adopt secondary structural motifs, such as disulfide-bonded segments, while maintaining a secretion-competent state. Here, we used the endogenous and closely spaced cysteine residues of the plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli to investigate the effect of disulfide bond-induced folding on translocation of an autotransporter passenger domain. We reveal that rigid structural elements within disulfide-bonded segments are resistant to autotransporter-mediated secretion. We define the size limit of disulfide-bonded segments tolerated by the autotransporter system demonstrating that, when present, cysteine pairs are intrinsically closely spaced to prevent congestion of the translocator pore by large disulfide-bonded regions. These latter data strongly support the hairpin mode of autotransporter biogenesis.