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Methods in Molecular Biology (Clifton,... 2023Primary cultures of bovine chromaffin cells are considered a good model to evaluate potential neuroprotective compounds for two major reasons: (i) they share many common...
Primary cultures of bovine chromaffin cells are considered a good model to evaluate potential neuroprotective compounds for two major reasons: (i) they share many common features to neurons as they synthesize, store, and release neurotransmitters; they are excitable cells that express voltage-dependent calcium, potassium, and sodium channels; they express different neuronal receptor subtypes; and (ii) they can be easily cultured in high quantities from adult animals; as adult para-neurons, they can be used to reproduce different neurodegenerative-like cytotoxicity models. In this chapter, we describe protocols to mimic calcium overload (veratridine and thapsigargin) and oxidative stress (rotenone plus oligomycin-A and 6-hydroxydopamine) to evaluate potential neuroprotective compounds.
Topics: Animals; Calcium; Cattle; Cells, Cultured; Chromaffin Cells; Neuroprotective Agents; Neurotransmitter Agents; Oligomycins; Oxidopamine; Potassium; Rotenone; Sodium Channels; Thapsigargin; Veratridine
PubMed: 36205906
DOI: 10.1007/978-1-0716-2671-9_24 -
Nature Communications Jun 2024Veratramine and cyclopamine, two of the most representative members of the isosteroidal alkaloids, are valuable molecules in agricultural and medicinal chemistry. While...
Veratramine and cyclopamine, two of the most representative members of the isosteroidal alkaloids, are valuable molecules in agricultural and medicinal chemistry. While plant extraction of these compounds suffers from uncertain supply, efficient chemical synthesis approaches are in high demand. Here, we present concise, divergent, and scalable syntheses of veratramine and cyclopamine with 11% and 6.2% overall yield, respectively, from inexpensive dehydro-epi-androsterone. Our synthesis readily provides gram quantities of both target natural products by utilizing a biomimetic rearrangement to form the C-nor-D-homo steroid core and a stereoselective reductive coupling/(bis-)cyclization sequence to establish the (E)/F-ring moiety.
Topics: Veratrum Alkaloids; Stereoisomerism; Cyclization; Biological Products; Molecular Structure
PubMed: 38909052
DOI: 10.1038/s41467-024-49748-2 -
Biomedical Chromatography : BMC Sep 2019The prominent stromal compartment surrounds pancreatic ductal adenocarcinoma and protects the tumor cells from chemo- or radiotherapy. We hypothesized that our nano...
Development and validation of a rapid and sensitive UPLC-MS/MS assay for simultaneous quantification of paclitaxel and cyclopamine in mouse whole blood and tissue samples.
The prominent stromal compartment surrounds pancreatic ductal adenocarcinoma and protects the tumor cells from chemo- or radiotherapy. We hypothesized that our nano formulation carrying cyclopamine (CPA, stroma modulator) and paclitaxel (PTX, antitumor agent) could increase the permeation of PTX through the stromal compartment and improve the intratumoral delivery of PTX. In the present study a sensitive, reliable UPLC-MS/MS method was developed and validated to quantify PTX and CPA simultaneously in mouse whole blood, pancreas, liver and spleen samples. Docetaxel was used as the internal standard. The method demonstrated a linear range of 0.5-2000 ng/mL for whole blood and tissue homogenates for both PTX and CPA. The accuracy and precision of the assay were all within ±15%. Matrix effects for both analytes were within 15%. Recoveries from whole blood, liver, spleen and pancreas homogenates were 92.7-105.2% for PTX and 72.8-99.7% for CPA. The stability was within ±15% in all test biomatrices. The validated method met the acceptance criteria according to US Food and Drug Administration regulatory guidelines. The method was successfully applied to support a pharmacokinetic and biodistribution study for PTX and CPA in mice biomatrices.
Topics: Animals; Chromatography, High Pressure Liquid; Limit of Detection; Linear Models; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms, Experimental; Paclitaxel; Pancreatic Neoplasms; Reproducibility of Results; Tandem Mass Spectrometry; Tissue Distribution; Veratrum Alkaloids
PubMed: 30805953
DOI: 10.1002/bmc.4518 -
Circulation. Genomic and Precision... Feb 2020Variants in ion channel genes have classically been studied in low throughput by patch clamping. Deep mutational scanning is a complementary approach that can...
BACKGROUND
Variants in ion channel genes have classically been studied in low throughput by patch clamping. Deep mutational scanning is a complementary approach that can simultaneously assess function of thousands of variants.
METHODS
We have developed and validated a method to perform a deep mutational scan of variants in , which encodes the major voltage-gated sodium channel in the heart. We created a library of nearly all possible variants in a 36 base region of in the S4 voltage sensor of domain IV and stably integrated the library into HEK293T cells.
RESULTS
In preliminary experiments, challenge with 3 drugs (veratridine, brevetoxin, and ouabain) could discriminate wild-type channels from gain- and loss-of-function pathogenic variants. High-throughput sequencing of the pre- and postdrug challenge pools was used to count the prevalence of each variant and identify variants with abnormal function. The deep mutational scan scores identified 40 putative gain-of-function and 33 putative loss-of-function variants. For 8 of 9 variants, patch clamping data were consistent with the scores.
CONCLUSIONS
These experiments demonstrate the accuracy of a high-throughput in vitro scan of variant function, which can be used to identify deleterious variants in and other ion channel genes.
Topics: Cardiotonic Agents; DNA Mutational Analysis; HEK293 Cells; Humans; Marine Toxins; Mutation; NAV1.5 Voltage-Gated Sodium Channel; Ouabain; Oxocins; Pharmacogenomic Testing; Veratridine
PubMed: 31928070
DOI: 10.1161/CIRCGEN.119.002786 -
Fitoterapia Sep 2019Veratrum californicum is a rich source of steroidal alkaloids, many of which have proven to be antagonists of the Hedgehog (Hh) signaling pathway that becomes aberrant...
Veratrum californicum is a rich source of steroidal alkaloids, many of which have proven to be antagonists of the Hedgehog (Hh) signaling pathway that becomes aberrant in over twenty types of cancer. These alkaloids first became known in the 1950's due to their teratogenic properties, which resulted in newborn and fetal lambs developing cyclopia as a result of pregnant ewes consuming Veratrum californicum. It was discovered that the alkaloids in V. californicum were concentrated in the root and rhizome of the plant with much lower amounts of the most active alkaloid, cyclopamine, present in the aerial plant, especially in the late growth season. Inspired by the limitations in analytical instrumentation and methods available to researchers at the time of the original investigation, we have used state-of-the-art instrumentation and modern analytical methods to quantitate four steroidal alkaloids based on study parameters including plant part, harvest location, and growth stage. The results of the current inquiry detail differences in alkaloid composition based on the study parameters, provide a detailed assessment for alkaloids that have been characterized previously (cyclopamine, veratramine, muldamine and isorubijervine), and identify at least six alkaloids that have not been previously characterized. This study provides insight into optimal harvest time, plant growth stage, harvest location, and plant part required to isolate, yet to be characterized, alkaloids of interest for exploration as Hh pathway antagonists with desirable medicinal properties.
Topics: Alkaloids; Hedgehog Proteins; Idaho; Molecular Structure; Phytochemicals; Plant Components, Aerial; Rhizome; Seasons; Steroids; Veratrum; Veratrum Alkaloids
PubMed: 31381957
DOI: 10.1016/j.fitote.2019.104281 -
Journal of Pineal Research Oct 2017Melatonin, which is synthesized in the pineal gland and peripheral reproductive organs, has antioxidant properties and regulates physiological processes. It is well...
Melatonin, which is synthesized in the pineal gland and peripheral reproductive organs, has antioxidant properties and regulates physiological processes. It is well known that melatonin affects in vitro maturation (IVM) of oocytes and embryonic development in many species. However, beneficial effects of melatonin on IVM have been explained mainly by indirect antioxidant effects and little information is available on the underlying mechanism by which melatonin directly acts on porcine cumulus oocyte complexes (COCs). Sonic hedgehog (Shh) signaling is important for follicle development, oocyte maturation, and embryo development, and there may be a relationship between melatonin and Shh signaling. To examine this, we designed three groups: (i) control, (ii) melatonin (10 mol/L), and (iii) melatonin with cyclopamine (2 μmol/L; Shh signaling inhibitor). The aim of this study was to investigate the effects of these agents on cumulus expansion, oocyte maturation, embryo development after parthenogenetic activation (PA), gene expression in cumulus cells, oocytes and blastocysts, and protein expression in COCs. Melatonin significantly increased the proportion of COCs exhibiting complete cumulus expansion (degree 4), PA blastocyst formation rates, and total cell numbers, which were inhibited by addition of cyclopamine. Simultaneously, the expression of cumulus expansion-related genes (Ptgs1, Ptgs2, and Has2) and Shh signaling-related genes (Shh, Pthc1, Smo, and Gli1) and proteins (Ptch1, Smo, and Gli1) in cumulus cells was upregulated in the melatonin-treated group, and these effects were also inhibited by cyclopamine. In conclusion, our results suggest that Shh signaling mediates effects of melatonin to improve porcine cumulus expansion and subsequent embryo development.
Topics: Animals; Antioxidants; Drug Evaluation, Preclinical; Embryonic Development; Female; Hedgehog Proteins; In Vitro Oocyte Maturation Techniques; Melatonin; Oocytes; Receptors, Melatonin; Signal Transduction; Swine; Veratrum Alkaloids
PubMed: 28512846
DOI: 10.1111/jpi.12424 -
Cardiovascular Toxicology Apr 2019Ivabradine has recently been demonstrated to have antiarrhythmic properties in atrial fibrillation. The aim of the present study was to assess the electrophysiologic...
Ivabradine has recently been demonstrated to have antiarrhythmic properties in atrial fibrillation. The aim of the present study was to assess the electrophysiologic profile of ivabradine in an experimental whole-heart model of long-QT-syndrome. In 12 isolated rabbit hearts long-QT-2-syndrome (LQT2) was simulated by infusion of D,L-sotalol (100 µM). 12 rabbit hearts were treated with veratridine (0.5 µM) to mimic long-QT-3-syndrome (LQT3). Sotalol induced a significant prolongation of QT-interval (+ 40 ms, p < 0.01) and action potential duration (APD, + 20 ms, p < 0.01). Similar results were obtained in veratridine-treated hearts (QT-interval: +52 ms, p < 0.01; APD: + 41 ms, p < 0.01). Of note, both sotalol (+ 26 ms, p < 0.01) and veratridine (+ 42 ms, p < 0.01) significantly increased spatial dispersion of repolarisation. Additional infusion of ivabradine (5 µM) did not change these parameters in sotalol-pretreated hearts but resulted in a further significant increase of QT-interval (+ 26 ms, p < 0.05) and APD (+ 49 ms, p < 0.05) in veratridine-treated hearts. Lowering of potassium concentration in bradycardic AV-blocked hearts resulted in the occurrence of early afterdepolarizations (EAD) or polymorphic ventricular tachycardias (VT) resembling torsade de pointes in 6 of 12 sotalol-treated hearts (56 episodes) and 6 of 12 veratridine-treated hearts (73 episodes). Additional infusion of ivabradine increased occurrence of polymorphic VT. Ivabradine treatment resulted in occurrence of EAD and polymorphic VT in 9 of 12 sotalol-treated hearts (212 episodes), and 8 of 12 veratridine-treated hearts (155 episodes). Treatment with ivabradine in experimental models of LQT2 and LQT3 increases proarrhythmia. A distinct interaction with potassium currents most likely represents a major underlying mechanism. These results imply that ivabradine should be employed with caution in the presence of QT-prolongation.
Topics: Action Potentials; Animals; Anti-Arrhythmia Agents; Heart Conduction System; Heart Rate; Isolated Heart Preparation; Ivabradine; Long QT Syndrome; Potassium; Rabbits; Risk Assessment; Sotalol; Tachycardia, Ventricular; Time Factors; Veratridine
PubMed: 30238354
DOI: 10.1007/s12012-018-9482-y -
The Turkish Journal of Gastroenterology... Mar 2020To investigate the effect and the possible mechanism of lanthanum citrate on the proliferation and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721...
BACKGROUND/AIMS
To investigate the effect and the possible mechanism of lanthanum citrate on the proliferation and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 through the Hedgehog (Hh) signaling pathway.
MATERIALS AND METHODS
Different concentrations of lanthanum citrate and KAAD-cyclopamine (the Hh signaling pathway representative inhibitor) were used to treat SMMC-7721 cells. Cell proliferation was detected using Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assays. Cell apoptosis was detected using flow cytometry analysis of Annexin V-FITC/ propidium iodide (PI). The protein expressions of regulatory genes, such as cell cycle protein D1 (CyclinD1), cyclin-dependent kinase inhibitor 1 (p21), cysteinyl aspartate specific proteinase 3 (Caspase-3), B-cell lymphoma-2 (Bcl-2), glioma-associated oncogene homolog 1 (Gli1), and sonic hedgehog (Shh) were quantified using Western blot assays. The mRNA expressions of Gli1 and Shh were tested using quantitative real-time polymerase chain reaction (qRT-PCR) assays and the protein expressions of Gli1 and Shh were determined using immunofluorescence assays.
RESULTS
The Annexin V-FITC and PI double staining results revealed that the 0.1 mM lanthanum citrate group and the 15 µM KAAD-cyclopamine group had both increased the apoptosis rate of SMMC-7721 cells. Both lanthanum citrate and KAAD-cyclopamine downregulated the protein expressions of CyclinD1, Bcl-2, Gli1, and Shh and upregulated the protein expressions of p21 and Caspase-3. Additionally, the immunofluorescence results revealed that the protein expressions of Gli1 and Shh were significantly decreased in both the lanthanum citrate group and the KAAD-cyclopamine group compared to the control group.
CONCLUSION
Lanthanum citrate inhibits proliferation and promotes apoptosis in HCC SMMC-7721 cells by suppressing the Hh signaling pathway.
Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cinnamates; Citrates; Hedgehog Proteins; Humans; Liver Neoplasms; Signal Transduction; Veratrum Alkaloids
PubMed: 32343239
DOI: 10.5152/tjg.2020.18800 -
Bioorganic & Medicinal Chemistry Jun 2021Constitutive activation of Hedgehog (Hh) pathway is intimately related with the occurrence and development of several malignancies, such as medulloblastoma (MB) and...
Constitutive activation of Hedgehog (Hh) pathway is intimately related with the occurrence and development of several malignancies, such as medulloblastoma (MB) and other tumors. Therefore, small molecular inhibitors of Hh pathway are urgently needed. In this study, three new steroidal alkaloids, ⊿ (20R, 24R) 23-oxo-24-methylsolacongetidine, ⊿ (20S, 24R) 23-oxo-24-methylsolacongetidine and veralinine 3-O-α-l-rhamnopyranosyl-(1 → 2)-β-D-glucopyranoside, together with six known alkaloids, 20-epi-verazine, verazine, protoverine 15-(l)-2'-methylbutyrate, jervine, veramarine and β1-chaconine, were isolated and determined from Veratrum grandiflorum Loes. The dual-luciferase bioassay indicated that all compounds exhibited significant inhibitions of Hh pathway with IC values of 0.72-14.31 μM against Shh-LIGHT 2 cells. To determine whether these Hh pathway inhibitors act with the Smoothened (Smo) protein, which is an important oncoprotein and target for this pathway, BODIPY-cyclopamine (BC) competitive binding assay was preferentially performed. Compared with BC alone, all compounds obviously reduced the fluorescence intensities of BC binding with Smo in Smo-overexpression HEK293T cells through fluorescence microscope and flow cytometer. By directly interacting with Smo, it revealed that they were actually novel natural Smo inhibitors. Then, their anti-tumor effects were investigated against the human MB cell line DAOY, which is a typical pediatric brain tumor cells line with highly expressed Hh pathway. Interestingly, most of compounds had slight proliferation inhibitions on DAOY cells after treatment for 24 h same as vismodegib, while β1-chaconine showed the strongest inhibitory effect on the growth of DAOY with IC value of 5.35 μM. In conclusion, our studies valuably provide several novel natural Smo inhibitors for potential targeting treatment of Hh-dependent tumors.
Topics: Alkaloids; Cell Line, Tumor; Cell Proliferation; HEK293 Cells; Humans; Medulloblastoma; Molecular Structure; Smoothened Receptor; Spectrum Analysis; Steroids; Veratrum
PubMed: 33910157
DOI: 10.1016/j.bmc.2021.116166 -
Clinical Toxicology (Philadelphia, Pa.) Sep 2018Steroidal alkaloids are found in plants of the genus Veratrum. Their toxicity manifests as gastrointestinal symptoms followed by a Bezold-Jarisch reflex: hypopnea,...
INTRODUCTION
Steroidal alkaloids are found in plants of the genus Veratrum. Their toxicity manifests as gastrointestinal symptoms followed by a Bezold-Jarisch reflex: hypopnea, hypotension, and bradycardia. Some Veratrum steroidal alkaloids are also teratogens interfering with the hedgehog-2 signaling pathway, which causes cyclopsia and holoprosencephaly. We present a case of accidental poisoning from Veratrum parviflorum mistaken for the edible Allium tricoccum (ramps, wild leek).
CASE HISTORY
A 27-year-old man and his 25-year-old wife presented to the emergency department with nausea, vomiting, hypotension, and bradycardia after foraging and ingesting plants that they believed to be a local native species of wild leek.
METHODS
We collected and analyzed the implicated fresh plant material and both patients' serum/plasma. We used liquid chromatography-mass spectroscopy and high-resolution electrospray ionization time of flight tandem mass spectrometry to extract and characterize steroidal alkaloids from the foraged plant and patients' serum.
RESULTS
Our V. parviflorum samples contained verazine, veratramine, veratridine, and cyclopamine.
DISCUSSION
Steroidal alkaloids have been previously isolated from Veratrum viride and Veratrum album and toxicity has been reported mainly from V. album species.
CONCLUSION
V. parviflorum toxicity manifests with gastrointestinal and cardiac symptoms. Treatment is symptomatic and supportive as with previous case reports of toxicity with other Veratrum species.
Topics: Adult; Antiemetics; Female; Gastrointestinal Diseases; Georgia; Humans; Male; Plant Poisoning; Treatment Outcome; Veratrum; Veratrum Alkaloids; Vomiting
PubMed: 29490507
DOI: 10.1080/15563650.2018.1442007