Heart-Cutting Bidimensional Liquid Chromatography for the Simultaneous Analysis of Veterinary Drugs Residues and Nucleotide Monophosphates in Sheep's Milk.

Sheep's milk is a significant source of nucleotide monophosphates (NMPs) but can also contain undesirable residues from veterinary drugs, posing a potential human health risk. This study introduces a novel application of two-dimensional liquid chromatography (2D-LC), in heart-cutting mode, for the simultaneous determination of nucleotides and veterinary drug residues in sheep's milk. 2D-LC allows for the separation of these compounds in a single chromatographic run despite their differing physicochemical properties. The proposed method separates six veterinary drug residues and five NMPs in a single injection. The compounds were separated using a C18 reversed-phase column in the first dimension and a Primesep SB analytical column in the second dimension. The method performance was evaluated in terms of linearity range, detection and quantification limits, matrix effects, precision, and accuracy. The results demonstrated good linearity and sensitivity, with quantification limits allowing for the quantification of veterinary drugs at the maximum residue level and nucleotides at typical levels found in milk samples. The method has been successfully applied to the analysis of sheep's milk samples acquired from local supermarkets, with recoveries within a range of 70-119% and 82-117% for veterinary residues and NMPs, respectively.

Electrophysiological Studies Revealed CaM1-Mediated Regulation of the Arabidopsis Calcium Channel CNGC12.

The Arabidopsis cyclic nucleotide-gated channel (CNGC) family consists of 20 members, which have been reported to participate in various physiological processes, such as pathogen defense, development, and thermotolerance. Although CNGC11 and CNGC12 have been identified a decade ago and their role in programmed cell death is well studied, their precise channel regulation has not been studied electrophysiologically. Here, we determined the channel activities of CNGC11 and CNGC12 utilizing the two-electrode voltage-clamp technique in the Xenopus laevis oocyte heterologous expression system. Our results suggest that CNGC12 but not CNGC11 functions as an active calcium channel. Furthermore, the cyclic nucleotide monophosphates (cNMPs) did not affect the activities of CNGC11 nor CNGC12 in Xenopus oocytes. Interestingly, while the activity of CNGC11 was not affected by co-expression with calmodulin (CaM), the activity of CNGC12 was significantly enhanced when CaM1 was co-expressed in oocytes. This study reveals that the channel activities and the mechanisms of regulation by CaM are different between CNGC11 and CNGC12.

Direct Determination of Six Cytokinin Nucleotide Monophosphates in Coconut Flesh by Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry.

Coconut contains many uncharacterized cytokinins that have important physiological effects in plants and humans. In this work, a method based on liquid chromatography-tandem mass spectrometry was developed for identification and quantification of six cytokinin nucleotide monophosphates in coconut flesh. Excellent separation was achieved using a low-coverage C18 bonded-phase column with an acidic mobile phase, which greatly improved the retention of target compounds. To enable high-throughput analysis, a single-step solid-phase extraction using mixed-mode anion-exchange cartridges was employed for sample preparation. This proved to be an effective method to minimize matrix effects and ensure high selectivity. The limits of detection varied from 0.06 to 0.3 ng/mL, and the limits of quantification ranged from 0.2 to 1.0 ng/mL. The linearity was statistically verified over 2 orders of magnitude, giving a coefficient of determination (R2) greater than 0.9981. The mean recoveries were from 81 to 108%; the intraday precision (n = 6) was less than 11%; and the interday precision (n = 11) was within 14%. The developed method was applied to the determination of cytokinin nucleotide monophosphates in coconut flesh samples, and four of them were successfully identified and quantified. The results showed that trans-zeatin riboside-5'-monophosphate was the dominant cytokinin, with a concentration of 2.7-34.2 ng/g, followed by N6-isopentenyladenosine-5'-monophosphate (≤12.9 ng/g), while the concentrations of cis-zeatin riboside-5'-monophosphate and dihydrozeatin riboside-5'-monophosphate were less than 2.2 and 4.9 ng/g, respectively.

Cyclic Nucleotide Monophosphates in Plants and Plant Signaling.

Cyclic nucleotide monophosphates (cNMPs) and the enzymes that can generate them are of increasing interest in the plant sciences. Arguably, the major recent advance came with the release of the complete Arabidopsis thaliana genome that has enabled the systematic search for adenylate (ACs) or guanylate cyclases (GCs) and did eventually lead to the discovery of a number of GCs in higher plants. Many of these proteins have complex domain architectures with AC or GC centers moonlighting within cytosolic kinase domains. Recent reports indicated the presence of not just the canonical cNMPs (i.e., cAMP and cGMP), but also the noncanonical cCMP, cUMP, cIMP, and cdTMP in plant tissues, and this raises several questions. Firstly, what are the functions of these cNMPs, and, secondly, which enzymes can convert the substrate triphosphates into the respective noncanonical cNMPs? The first question is addressed here by comparing the reactive oxygen species (ROS) response of cAMP and cGMP to that elicited by the noncanonical cCMP or cIMP. The results show that particularly cIMP can induce significant ROS production. To answer, at least in part, the second question, we have evaluated homology models of experimentally confirmed plant GCs probing the substrate specificity by molecular docking simulations to determine if they can conceivably catalytically convert substrates other than ATP or GTP. In summary, molecular modeling and substrate docking simulations can contribute to the evaluation of cyclases for noncanonical cyclic mononucleotides and thereby further our understanding of the molecular mechanism that underlie cNMP-dependent signaling in planta.

Analysis of free nucleotide monophosphates in human milk and effect of pasteurisation or high-pressure processing on their contents by capillary electrophoresis coupled to mass spectrometry.

A simple, efficient and green analytical method for the determination of free nucleotide monophosphates in human milk is proposed. It involves centrifugal ultrafiltration (CUF) as sample treatment and capillary electrophoresis-electrospray mass spectrometry (CE-ESI-MS) for separation and simultaneous quantification. The optimised method, applied to the analysis of human milk samples, included their dilution (1:5) with water followed by CUF treatment. No matrix effects were found. The method provided limits of detection between 0.08 and 0.13 µg mL(-1) and limits of quantification between 0.26 and 0.43 µg mL(-1). The intralaboratory repeatability and reproducibility afforded relative standard deviation values lower than 10%. The method was applied to the study of the effects of Holder pasteurisation and high-pressure processing on the nucleotide contents in samples from a human milk bank. The results showed concentration values between 0.5 and 10 µg mL(-1), with higher concentrations for the samples treated by pasteurisation. The effect of freezing time on the content of nucleotides was also assessed.

Measurement of 2',3'-cyclic nucleotides by liquid chromatography-tandem mass spectrometry in cells.

Recently, the occurrence of 2',3'-cyclic nucleoside monophosphates (2',3'-cNMPs) in addition to 3',5'-cNMPs in mammalian tissues was reported. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of four 2',3'-cyclic nucleotides, i.e., 2',3'-cAMP, 2',3'-cCMP, 2',3'-cGMP, 2',3'-cUMP, in cell samples. Chromatographic separation was achieved using a Zorbax eclipse XCB-C18 (50 mm×4.6 mm; 1.8 µm column; Agilent) connected to a QTRAP5500 system (AB Sciex) operating in positive ionization mode. Calibration curves were constructed in the range 0.41 fmol/µL to 1666.6 fmol/µL for 2',3'-cAMP, 2',3'-cCMP, and 2',3'-cGMP, and 3.3-1666.6 fmol/µL for 2',3'-cUMP, respectively, showing squared correlation coefficients >0.9992. Accuracy and inter- and intra-day precision lay within the required ranges of <20% for LLOQ and <15% for higher concentration levels. The method was applied to the analysis of nucleotides in two different cell lines (Hek293T and HuT-78).

Evidence for the role of hydrophobic forces on the interactions of nucleotide-monophosphates with cationic liposomes.

In this work, the interaction of nucleotide-monophosphates (NMPs) with unilamellar liposomes made of 1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP) and 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE) was investigated. Here, we demonstrate how adsorption is affected by the type of nucleotide-monophosphate. Dynamic light scattering (DLS) results revealed, for each NMP, that a distinguishable concentration exists at which a significant growth of the aggregates occurs. Adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) have shown a higher propensity to induce liposome aggregation process and in particular GMP appears to be the most effective. From ζ-potential experiments we found that liposomes loaded with purine based nucleotides (AMP and GMP) are able to decrease the ζ-potential values to a greater extent in comparison with the pyrimidine based nucleotides thimydine 5'-monophosphate (TMP) and uridine 5'-monophosphate (UMP). Moreover, a careful analysis of nucleotide-liposome interactions revealed that nucleotides have different capacity to induce the formation of nucleotide-liposome complexes, and purine based nucleotides have higher affinities with lipid membranes. On the whole, the data emphasize that the mechanisms driving the interactions between liposomes and NMPs are also influenced by the existence of hydrophobic forces.