Puromycin reveals a distinct conformation of neuronal ribosomes.

Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It was postulated that blocking the translocation of the ribosome with emetine could retain the puromycylated peptide on the ribosome, but evidence against this has recently been published [Hobson et al., Elife 9, e60048 (2020); and Enam et al., Elife 9, e60303 (2020)]. In neurons, puromycylated nascent chains remain in the ribosome even in the absence of emetine, yet direct evidence for this has been lacking. Using biochemistry and cryoelectron microscopy, we show that the puromycylated peptides remain in the ribosome exit channel in the large subunit in a subset of neuronal ribosomes stalled in the hybrid state. These results validate previous experiments to localize stalled polysomes in neurons and provide insight into how neuronal ribosomes are stalled. Moreover, in these hybrid-state neuronal ribosomes, anisomycin, which usually blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains that are stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide insights into the stalling mechanisms of neuronal ribosomes and suggest that puromycylated peptides can be used to reveal subcellular sites of hybrid-state stalled ribosomes in neurons.

The Giardia lamblia ribosome structure reveals divergence in several biological pathways and the mode of emetine function.

Giardia lamblia is a deeply branching protist and a human pathogen. Its unusual biology presents the opportunity to explore conserved and fundamental molecular mechanisms. We determined the structure of the G. lamblia 80S ribosome bound to tRNA, mRNA, and the antibiotic emetine by cryo-electron microscopy, to an overall resolution of 2.49 Å. The structure reveals rapidly evolving protein and nucleotide regions, differences in the peptide exit tunnel, and likely altered ribosome quality control pathways. Examination of translation initiation factor binding sites suggests these interactions are conserved despite a divergent initiation mechanism. Highlighting the potential of G. lamblia to resolve conserved biological principles; our structure reveals the interactions of the translation inhibitor emetine with the ribosome and mRNA, thus providing insight into the mechanism of action for this widely used antibiotic. Our work defines key questions in G. lamblia and motivates future experiments to explore the diversity of eukaryotic gene regulation.

Sweeping of hydrophobic amines under inhomogeneous electric field and low surfactant concentration in micellar electrokinetic chromatography.

The influence of sample matrix on sample sweeping in MEKC was examined in the presented manuscript. Significant focusing effect was observed for relatively hydrophobic cationic compounds (emetine, strychnine and quinine) using high ionic strength sample matrix (900 mM H3 PO4 /720 mM Tris) which conductivity was about ninefold higher than utilized BGE. Moreover, the results were obtained using BGE composed of comparatively low surfactant concentration (10 mM SDS) and 40 mM H3 PO4 /32 mM Tris buffer solution. About 200 to 300-fold preconcentration of analytes was reached with the presented method. Basing on experimental results and computer simulation using Simul5 software, hypothetical mechanism of observed phenomenon was proposed.

Molecular phylogeography of Carapichea ipecacuanha, an amphitropical shrub that occurs in the understory of both semideciduous and evergreen forests.

The medicinal shrub Carapichea ipecacuanha (ipecac) is an amphitropic species with three disjunct areas of distribution. In the Brazilian Atlantic and Amazonian ranges, the species was associated mostly with the understory of seasonal semideciduous forests, whereas in the Central American-Colombian range, the species occurred in the understory of moist evergreen forests. We examined the phylogeographic structure of ipecac using chloroplast trnT-trnL and nuclear internal transcribed spacer (ITS) sequences from 120 and 46 specimens, respectively. To complement existing data on root alkaloid profiles, we used high-performance liquid chromatography to assess the levels of emetine and cephaeline in 33 specimens from the two Brazilian ranges. The three ranges shared neither nuclear nor chloroplast haplotypes. The phylogeographic structures showed an uneven distribution of genetic diversity, sharp breaks and high levels of genetic differentiation among ranges. Our results suggest that the extant populations are descendents of at least four distinct ancestral lineages. The Atlantic ipecacs showed higher levels of genetic diversity than ipecacs from the other two ranges; it is likely that they derive from two ancestral lineages, with long-term persistence in that region. The Amazonian ipecacs were monomorphic with respect to the ITS and cpDNA sequences, which supports the view that there was a recent expansion from a single parental source after a strong genetic bottleneck. The existence of a fourth distinct lineage is apparent from the high levels of genetic and chemical differentiation that we identified in the Central American-Columbian ipecacs.

Ipecac syrup abuse, morbidity, and mortality: isn't it time to repeal its over-the-counter status?

PURPOSE: To review and address the abuse of ipecac, describing its epidemiology, toxicity, clinical characteristics, and laboratory assessment. METHODS: A Medline search (1980-2003) for Ipecac abuse and Ipecac toxicity, n = 34. RESULTS: Ipecac abuse occurs predominantly among adolescent and young adult females who are either experimenting with purging or have an eating disorder. Psychiatric comorbidity is common. Death can occur and is usually of cardiac origin. Morbidity includes myocarditis with arrhythmias, myositis, gastroesophageal pathology, including Mallory Weiss tears, diarrhea, and metabolic abnormalities (alkalosis, hypokalemia, dehydration). The injuries can reverse with cessation of ipecac use. A high index of suspicion is needed for early detection. Classic findings are abnormal EKG and echocardiography and/or elevation of muscle enzymes (CPK, adolase). Emetine, the alkaloid in ipecac, can be confirmed in serum, urine, and tissue by high performance liquid chromatography. CONCLUSIONS: Ipecac abuse is dangerous, even deadly. However, if abuse is discontinued, cardiac and muscle damage tends to reverse. Were ipecac syrup to remain an over- the-counter medication, or become a prescription medication, more stringent warning labels ought to be included and further education be provided about its toxicity and potential for abuse. Removing ipecac from the over-the-counter category would best eliminate its potential for abuse.

Determination of chlorpheniramine maleate and tincture ipecac in dosage form by liquid chromatography with ultraviolet detection.

A procedure was developed and validated for measuring chlorpheniramine maleate and tincture ipecac (as emetine hydrochloride) by reversed-phase liquid chromatography with methanol-10 mM sodium heptanesulfonate (20 + 30) as the mobile phase; the pH was adjusted to 4 with acetic acid, and the flow rate was at 1.5 mL/min, with ultraviolet detection at 254 nm. Propyl paraben was used as the internal standard. The standard curves were linear (r = 0.998 and 0.9998) for both chlorpheniramine maleate and emetine hydrochloride over the ranges of 5-100 and 0.1-40 microg/mL, respectively. The mean recoveries +/- standard deviation were 101.37 +/- 2.77% for chlorpheniramine maleate and 98.8 +/- 1.47% for emetine hydrochloride. The proposed method was applied to the determination of chlorpheniramine maleate alone in tablet and syrup dosage forms. The method also was applied to the determination of the emetine content of ipecac liquid extract and tincture ipecac; the results were compared with those of the method of the British Pharmacopoeia. The proposed method was applied successfully to the simultaneous determination of chlorpheniramine maleate and tincture ipecac, as emetine hydrochloride, in syrup dosage form. Both drugs and the internal standard were separated from all interfering components in < 5 min. The proposed method is simple, specific, and economical, when compared with other published methods that determine each component alone.

Separation and determination of emetine dithiocarbamate metal complexes by capillary electrophoresis with chemiluminescence detection of the tris(2,2'-bipyridine)-ruthenium(II) complex.

Emetine dithiocarbamate metal complexes, which were prepared from emetine, carbon disulfide, and metal(II), indicated large chemiluminescence intensities on tris(2,2'-bipyridine)-ruthenium(II) chemiluminescence. Capillary electrophoresis with chemiluminescence detection was developed for analyzing emetine and the metal complexes. After the optimization of various analytical conditions, the mixture of the Cu(II), Ni(II), and Co(II) complexes as a model sample was injected into the capillary electrophoresis system with chemiluminescence detection. They were successfully separated and detected with a detection limit of 50 nM.

Selective, stability-indicating assay of the major ipecacuanha alkaloids, emetine and cephaeline, in pharmaceutical preparations by high-performance liquid chromatography using spectrofluorimetric detection.

A selective high-performance liquid chromatographic procedure has been developed for the determination of the major Ipecacuanha alkaloids, emetine and cephaeline, in a number of linctus and pastille preparations. The reversed-phase chromatographic procedure uses an octadecyl-bonded column with a mobile phase of aqueous methanol containing an ion-pairing reagent. A spectrofluorimetric detector is used for increased sensitivity and selectivity. Sample preparation is simple, involving either straight dilution for linctus formulations or simple dissolutions for pastilles. The procedure has been shown to be stability-indicating. Validation studies, to show that the method is precise, accurate and rectilinear, have been carried out on four linctus formulations and two pastille formulations. The method has been used to determine both emetine and cephaeline at levels as low as 5 micrograms/g in formulations.

The application of chemical derivatization to clinical drug analysis.

The sensitivity and selectivity achievable in the analysis of drug substances from biological matrices is often limited by the physical and chemical properties of the analyte. These limitations are further exacerbated by the inherent reactivity of most drugs in biological systems (i.e., their propensity for undergoing biotransformation). One very powerful approach that has been taken to improve the quality of the analytical methodology is to alter the physico-chemical properties of the drug through chemical modification (derivatization) during some stage of the analytical sequence. This approach has been successfully applied to situations and has resulted in improved chemical stability, analytical selectivity and sensitivity. In most cases, drug analysis from biological fluids involves a chromatographic step; the derivatization reaction can be carried out either prior or subsequent to chromatography. In this paper, examples of the advantages (and limitations) offered by the introduction of a chemical derivatization step in clinical drug analysis will be presented. Specifically, focus will be placed on analysis of chemically-reactive antineoplastic agents and peptides/proteins. The latter represent an emerging class of drugs which present significant analytical challenges. The use of o-phthalaldehyde analogues offering improved derivative stability and increased sensitivity will be described.

Absorption of ipecac alkaloids in emergency patients.

Syrup of ipecac contains the nauseant alkaloids emetine and cephaeline. Although thousands of doses are given yearly, no data exist on the absorption of these alkaloids in man. We gave 30 mL of USP Syrup to ten adult patients. Blood and urine samples were obtained at approximately one-half and two hours after administration, and the entire volume of vomitus was saved. The samples were then analyzed for cephaeline and emetine by a high-performance liquid chromatographic (HPLC) assay developed in our laboratory. All patients vomited within 30 minutes, but the amounts of alkaloid regurgitated varied from 22 +/- 14% in six patients to 80 +/- 16% in the remaining four. Only six patients had emetine or cephaeline in their blood by two hours (range, 5 to 73 ng/mL), although ten patients had detectable concentrations of the alkaloids in their urine. Measured over two hours, no patient eliminated more than 0.5% of the dose by the urinary route. In our study ipecac was absorbed by all who received it; the extent of absorption varied widely, and elimination by the renal route was small.

Quantitative analysis of emetine and cephaeline by reversed-phase high performance liquid chromatography with fluorescence detection.

A versatile method for the quantitation of emetine and cephaeline in biological samples is described. Two milliliters of samples containing N-propylprocainamide as the internal standard are buffered to pH 9 and extracted with n-butyl chloride. After subsequent back extraction into 0.01 M hydrochloric acid, a portion of the acid layer is analyzed by reversed-phase high performance liquid chromatography with fluorescence detection. Routinely, the minimum level of detection for both drugs is 5 ng/mL and linearity is demonstrated from 5 to 2500 ng/mL.

Liquid chromatography of dansyl derivatives of some alkaloids and the application to the analysis of pharmaceuticals.

Derivatization of the alkaloids cephaeline, codeine, emetine, ephedrine, morphine, narcotine and others with dansyl chloride has been studied with the aim of developing a sensitive and specific liquid chromatographic method for these substances in complex pharmaceutical dosage forms. While codeine and narcotine do not react, the other compounds form completely substituted derivatives which possess maxima in their fluorescence emission spectra between 470 and 500 nm. The structure of the derivatives has been confirmed by nuclear magnetic resonance spectroscopy. The dansylated compounds have been separated by thin-layer chromatography and high-pressure liquid chromatography. The improved selectivity and sensitivity have permitted an analysis of these substances present in low concentrations in 10- 100-fold excesses of other drugs. Direct derivatization of syrups and aqueous slurries of capsules having a complex excipient and drug composition is feasible and time saving and serves as a pre-clean-up step. Detection limits are in the 1-10-ng range or better, depending on the efficiency of the detection device. The reproducibility of the method is limited by the derivatization step, but a relative standard deviation of less than 2% can be obtained. The analysis time for these pharmaceuticals may be reduced by at least one fifth of that required by conventional techniques.