Synthesis of S-(28a-homobetulin-28a-yl) thiophosphate, thiophosphonate, and thiophosphinate

<p>A concise synthesis of thiophosphate, phenylthiophosphonate, and diphenylthiophosphinate esters bearing a 28a-homolupane residue is reported. The new triterpenes were obtained from the readily available 3-<i>O</i>-acetylichopanol by a nucleophilic substitution of the corresponding mesylate with thiocyanate ion followed by a Michaelis–Arbuzov reaction. These results open the way to new lupane-type derivatives having a thiophosphorus moiety at the lupane core as potential anticancer compounds. Additionally, the cytotoxic activities of the new homolupane compounds were evaluated <i>in vitro</i>.</p

Simultaneous Determination of Selected Steroids with Neuroactive Effects in Human Serum by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

Neuroactive steroids are a group of steroid molecules that are involved in the regulation of functions of the nervous system. The nervous system is not only the site of their action, but their biosynthesis can also occur there. Neuroactive steroid levels depend not only on the physiological state of an individual (person’s sex, age, diurnal variation, etc.), but they are also affected by various pathological processes in the nervous system (some neurological and psychiatric diseases or injuries), and new knowledge can be gained by monitoring these processes. The aim of our research was to develop and validate a comprehensive method for the simultaneous determination of selected steroids with neuroactive effects in human serum. The developed method enables high throughput and a sensitive quantitative analysis of nine neuroactive steroid substances (pregnenolone, progesterone, 5α-dihydroprogesterone, allopregnanolone, testosterone, 5α-dihydrotestosterone, androstenedione, dehydroepiandrosterone, and epiandrosterone) in 150 μL of human serum by ultrahigh-performance liquid chromatography with tandem mass spectrometry. The correlation coefficients above 0.999 indicated that the developed analytical procedure was linear in the range of 0.90 nmol/L to 28.46 μmol/L in human serum. The accuracy and precision of the method for all analytes ranged from 83 to 118% and from 0.9 to 14.1%, respectively. This described method could contribute to a deeper understanding of the pathophysiology of various diseases. Similarly, it can also be helpful in the search for new biomarkers and diagnostic options or therapeutic approaches

Inhibition of CDK9 but not cell cycle dependent CDKs increases neutrophil apoptosis.

<p>(a) Isolated human neutrophils were incubated with NU6102 (filled triangle), R-roscovitine (filled square), or LGR1406 (filled diamond) at the concentrations shown. Apoptosis was determined after 15h by assessment of DiOC₆ uptake and flow cytometry, with DiOC₆ low cells taken as apoptotic. Data are mean ± s.d. for four separate experiments. * indicates p<0.05 for treatment versus control. (b) Neutrophils were incubated with flavopiridol for 15 h at the concentrations shown and apoptosis determined by assessment of DiOC₆ uptake. Data are mean ± s.d. for three separate experiments. * indicates p<0.05 for control versus flavopiridol treated cells. (c) Images of Giemsa stained neutrophils cultured in the absence (control) or presence of 100 nM flavopiridol for 15 h.</p

Human neutrophils express only cell cycle independent CDKs.

<p>Isolated human neutrophils (N) and promyelocytic HL60 cells (H) were assessed for expression of CDK proteins by western blotting (upper panel). Blots were scanned to determine the CDK:β-actin ratio by densitometry (lower panel; black bars  =  HL60, grey bars  =  neutrophils). Data are mean ± s.d. for three separate cell extracts.</p

Flavopiridol inhibits upregulation of Mcl-1 by GM-CSF.

<p>(a). Neutrophils were incubated with medium alone, or medium containing 100 nM flavopiridol in the absence (open bars) or presence of (filled bars) of 50 ng/ml GM-CSF. Live cells were detected by their ability to retain DiOC₆ and data are mean ± s.d. of 3 separate experiments. * indicates p<0.05 for control versus flavopiridol treated cells. (b). The same cells were extracted and measured for Mcl-1 content by western blotting. Blots were probed for β-actin to confirm equal loading.</p

CDK9 activity and cyclin T1 expression decrease as neutrophils age in culture.

<p>Isolated human neutrophils were cultured for 9h and (a) CDK9 protein expression and (b) enzymatic activity were determined and compared with freshly isolated cells (0h). For CDK9 activity protein was immunoprecipitated from neutrophils with a monoclonal anti-CDK9 antibody and activity determined by incorporation of [γ ³²P]-ATP into the CTD of RNA polymerase II. The phosphorylated peptide was isolated on an SDS-PAGE gel and excised for scintillation counting. Data are expressed as % of the value for control freshly isolated cells and are mean ± s.d. for three separate experiments. * indicates p<0.05. (c). Isolated neutrophils were cultured for 0 h or 9 h and expression of cyclin T1 determined by western blotting. Blots were probed with an antibody to β-actin to confirm equal protein loading. The blot shown is representative of 4 separate experiments performed. (d) Western blots of CDK9 expression relative to β-actin were quantitated using densitometry.</p

Conversion of 12-oxophytodienoic acid (OPDA) in leaves of <i>Arabidopsis thaliana</i> ecotype WS (wild type, A) and <i>opr3</i> (B).

<p>A, Leaves of 5-week-old WS plants grown under short-day conditions were floated on water (control, black bars) or on 50 μM OPDA for the time periods indicated. Levels of OPDA, jasmonic acid (JA) and JA-Ile are presented. B, Leaves of 5-week-old <i>opr3</i> plants grown under short-day conditions were floated on solutions containing different concentrations of OPDA for 30 min. Levels of OPDA (red bars), JA (blue bars) and JA-Ile (green bars) are presented. Contents of OPDA, JA and JA-Ile were determined by LC-MS according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162829#pone.0162829.ref030" target="_blank">30</a>]. Each value is represented by the mean of three independent biological replicates ± SD. Treatments and controls were pairwise compared by the Student’s t-test, *p≤0.05, **p≤0.01, ***p≤0.001.</p

Levels of OPDA (A), OPDA-Ile (B), JA (C) and JA-Ile (D) in seedlings of <i>Arabidopsis thaliana</i> WS, <i>opr3</i>, Col-0 and <i>jar1</i> after treatment with OPDA or OPDA-Ile.

<p>10-days-old seedlings grown in liquid culture were treated with 30 μM OPDA or 30 μM OPDA-Ile for 30 min (white bars). Two independent controls (black bars) were performed by treatment with bi-distilled H₂O containing 0.87% [v/v] and 0.56% [v/v] acetonitrile, respectively. Compounds were quantified according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162829#pone.0162829.ref024" target="_blank">24</a>]. Each value is represented by the mean of five independent biological replicates ± SD. Treatments and controls were pairwise compared by the Student’s t-test, *p≤0.05, **p≤0.01, ***p≤0.001.</p

Relative transcript levels of <i>GRX480</i> (A) and <i>ZAT10</i> (B) in seedlings of <i>Arabidopsis thaliana</i> WS, <i>opr3</i>, Col-0 and <i>jar1</i> after treatment with OPDA or OPDA-Ile.

<p>10-days-old seedlings grown in liquid culture were treated with 30 μM OPDA or 30 μM OPDA-Ile for 30 min (white bars). Two independent controls (black bars) were performed by treatment with bi-distilled H₂O containing 0.87% [v/v] and 0.56% [v/v] acetonitrile, respectively. Relative transcript levels were quantified by qRT-PCR using <i>AtPP2A</i> as reference. Each value is represented by the mean of three independent biological replicates ± SD. Treatments and controls were pairwise compared by the Student’s t-test, *p≤0.05, **p≤0.01, ***p≤0.001.</p

Expression profiles of genes involved in ethylene and ABA production and CK glycosylation and deglycosylation.

<p>Changes in expression were followed by qPCR in time intervals ranging from 30 minutes to 3 days in roots (<b>A</b>) and aerial part (<b>B</b>) of maize plantlets (6 to 8 days of development). <b>3MeOBAP</b> (black bars), <b>3MeOBA9THPP</b> (grey bars) and <b>3MeOBAP9G</b> (white bars) were applied in 1 μM concentration to the nutrient solution. All data are accomplished from three independent biological replicates run in at least two technical replicates. Genes for actin and elongation factor 1 were used as reference genes. Expression change due to control plants considered as statistically significant is indicated by asterisks (unpaired Student's <i>t</i> test with <i>P</i> ≤ 0.05). <b>ACS</b> – ethylene precursor synthase; <b>ACO</b> – ethylene release enzyme; <b>cZOGT</b> – cis-zeatin-<i>O</i>-glucosyltransferase; <b>bGLU</b> – <i>β</i>-glucosidase; <b>VP-14</b> – ABA biosynthetic gene. Graphs for <i>ACS</i> and <i>ACO</i> genes are arrayed from left to right side as abundance of particular gene decreases in given tissue.</p