Simultaneous high-performance liquid chromatographic analysis of flunitrazepam and four metabolites in serum

A high-performance liquid chromatographic method for the simultaneous determination of flunitrazepam and four metabolites, desmethylflunitrazepam (DMF), 7-aminodesmethylflunitrazepam (7-NH₂DMF), 7-aminoflunitrazepam (7-NH₂F) and 3-hydroxyflunitrazepam (3-OHF), in serum is described. The method involves a simple extraction from alkalinized plasma (pH 9.5) into diethyl ether-chloroform (80:20, v/v). Prazepam was used as an internal standard for the quantification of the five compounds. Separation was achieved with a 10 μm RSil CN column (300×3.9 mm I.D.). The detection wavelength was set at 242 nm. The limits of detection ranged from 2.5 to 5 μg/l with a limit of quantification of 10 μg/l for all analytes.     

Development of a one-step enzyme immunoassay for the quantitation of gibberellin A3 in barley seeds

The development of a sensitive and specific enzyme immunoassay for GA₃ is reported. This method was based on the use of peroxidase labelled GA₃ and immobilized antibodies. In order to obtain a rapid immunoassay, several steps of purification were analyzed to show their necessity. Barley seed extracts were assayed at different steps of purification to exhibit the effect of extract components on the assay. It was demonstrated that HPLC had to be performed when a selective quantitation of GA₃ was required. This assay allowed GA₃ to be measured with reproducibility as its unmethylated form and the quantitation of GA₃ in barley seeds with this enzyme immunoassay was correlated to a GC-MS method.Abbreviations GA₃ gibberellin A₃ - EIA enzyme immunoassay - DMF dimethylformamide - TEA tri(n)ethylamine - BSA bovine serum albumin - OVA ovalbumine - ECF ethylchloroformate - PB phosphate buffer     

Increase of cyclin B by overexpression of cystatin α

Degradation of cyclin B was effectively suppressed when cells were treated with ALLN (N-acetylleucylleucylnorleucinal) which inhibits proteasome, calpain and cysteine proteinase cathepsins. In order to examine which protease degrades cyclin B, the effect of a cathepsin inhibitor, cystatin α, was investigated. The cystatin α gene was inserted into an inducible expression vector, pMSG, and transfected into NIH3T3 mouse fibroblasts. The expression of cystatin α was induced effectively in the transfected cells after treatment with dexamethasone. Overexpression of cystatin α resulted in an increase of the amount of cyclin B, suggesting that cysteine proteinase cathepsins might be involved in the degradation of cyclin B.     

The role of lipid second messengers in aldosterone synthesis and secretion

Second messengers are small rapidly diffusing molecules or ions that relay signals between receptors and effector proteins to produce a physiological effect. Lipid messengers constitute one of the four major classes of second messengers. The hydrolysis of two main classes of lipids, glycerophospholipids and sphingolipids, generate parallel profiles of lipid second messengers: phosphatidic acid (PA), diacylglycerol (DAG), and lysophosphatidic acid versus ceramide, ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate, respectively. In this review, we examine the mechanisms by which these lipid second messengers modulate aldosterone production at multiple levels. Aldosterone is a mineralocorticoid hormone responsible for maintaining fluid volume, electrolyte balance, and blood pressure homeostasis. Primary aldosteronism is a frequent endocrine cause of secondary hypertension. A thorough understanding of the signaling events regulating aldosterone biosynthesis may lead to the identification of novel therapeutic targets. The cumulative evidence in this literature emphasizes the critical roles of PA, DAG, and sphingolipid metabolites in aldosterone synthesis and secretion. However, it also highlights the gaps in our knowledge, such as the preference for phospholipase D-generated PA or DAG, as well as the need for further investigation to elucidate the precise mechanisms by which these lipid second messengers regulate optimal aldosterone production.     

The antitumor agent 3-bromopyruvate has a short half-life at physiological conditions

Clinical research is currently exploring the validity of the anti-tumor candidate 3-bromopyruvate (3-BP) as a novel treatment for several types of cancer. However, recent publications have overlooked rarely-cited earlier work about the instability of 3-BP and its decay to 3-hydroxypyruvate (3-HP) which have obvious implications for its mechanism of action against tumors, how it is administered, and for precautions when preparing solutions of 3-BP. This study found the first-order decay rate of 3-BP at physiological temperature and pH has a half-life of only 77 min. Lower buffer pH decreases the decay rate, while choice of buffer and concentration do not affect it. A method for preparing more stable solutions is also reported.     

Identification of 3-deoxy-lyxo-2-heptulosaric acid in the core region of lipopolysaccharides from Rhizobiaceae

A 3-deoxy-2-heptulosaric acid (DHA), very probably with the lyxo-configuration, was identified in the R-core region of lipopolysaccharides from nodulating strains of Rhizobium leguminosarum, Rhizobium meliloti and from all three biovars of the phytopathogenic Agrobacterium tumefaciens. Its structure could be deduced from the fragmentation pattern of the corresponding alditol acetates obtained after reduction of the 2-keto and the 1.7-carboxy groups by sodium borohydride or sodium borodeuteride. DHA in lipopolysaccharide was not destroyed by periodate and is therefore not in a terminal position. Two DHA-containing oligosaccharides, namely glucosyl (1----4)-3-deoxy-2-heptulosaric acid and rhamnosyl-rhamnosyl-(1----5)-3-deoxy-2-heptulosaric acid could be tentatively identified by mass spectrometric methods amongst the products of mild acidic hydrolysis of lipopolysaccharides of Rhizobium leguminosarum strain 24. The two types of non-nodulating mutants of Rhizobium leguminosarum included in this study did not contain 3-deoxy-2-heptulosaric acid.     

Associations between manganese exposure and multiple immunological parameters in manganese-exposed workers healthy cohort

BackgroundManganese (Mn) ions play a crucial role in the immune response. The immunotoxicity of Mn is rarely reported compared with the neurotoxicity of Mn.ObjectivesThe purpose of this study was to investigate the associations between chronic Mn exposure and immunological parameters in occupational Mn-exposed workers.MethodsA total of 538 workers were selected from the follow-up of manganese-exposed workers healthy cohort (MEWHC) in 2017. We divided the workers into the low-exposure group and the high-exposure group by the cutoff of the manganese-time weighted average (Mn-TWA) setting at 0.15 mg/m³. We examined serum immunological parameters by the immunoturbidimetric method and leukocyte counts and ratios in blood routine. Then we used the generalized linear model analyses and spline analyses to explore the associations between external exposure of Mn and multiple immunological parameters adjusted for variables. Based on the epidemiological analyses, we used Elisa (enzyme-linked immune sorbent assay) to detect plasma complement C3 of Mn-exposed rats.ResultsIn male workers, the mean value of complement C3 was 1.20 ± 0.16 g/L in the high-exposure group, which was significantly lower as compared to the low-exposure group (1.25 ± 0.18 g/L, P = 0.023). The generalize linear models’ analyses showed that complement C3 value had a significantly negative association with external exposure of Mn included adjustment for variables (β = -0.04, P = 0.035). Moreover, in male rats, the high-exposure group also had a lower level of complement C3 compared with the low-exposure group (P < 0.001). None significant association was observed in immunological parameters among female workers and rats (all P > 0.05).ConclusionsMn exposure from inhalable dust was associated with decreased complement C3 among occupationally Mn-exposed male individuals but not in female workers, which was further confirmed by the rat model. Further research into the possible mechanism of C3 reduction is needed in the future.