Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene ...itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.
The safety and efficacy of kratom (Mitragyna speciosa) for treatment of pain is highly controversial. Kratom produces more than 40 structurally related alkaloids, but most studies have focused on ...just two of these, mitragynine and 7-hydroxymitragynine. Here, we profiled 53 commercial kratom products using untargeted LC-MS metabolomics, revealing two distinct chemotypes that contain different levels of the alkaloid speciofoline. Both chemotypes were confirmed with DNA barcoding to be M. speciosa. To evaluate the biological relevance of variable speciofoline levels in kratom, we compared the opioid receptor binding activity of speciofoline, mitragynine, and 7-hydroxymitragynine. Mitragynine and 7-hydroxymitragynine function as partial agonists of the human µ-opioid receptor, while speciofoline does not exhibit measurable binding affinity at the µ-, δ- or ƙ-opioid receptors. Importantly, mitragynine and 7-hydroxymitragynine demonstrate functional selectivity for G-protein signaling, with no measurable recruitment of β-arrestin. Overall, the study demonstrates the unique binding and functional profiles of the kratom alkaloids, suggesting potential utility for managing pain, but further studies are needed to follow up on these in vitro findings. All three kratom alkaloids tested inhibited select cytochrome P450 enzymes, suggesting a potential risk for adverse interactions when kratom is co-consumed with drugs metabolized by these enzymes.
We use a parallel frequency-domain optical coherence tomography (FDOCT) system to generate a scatter-mode image of the hamster cheek pouch epithelium. To our knowledge, this is the first optical ...coherence tomography (OCT) image of a biological sample obtained using a thermal light source in the frequency domain. The system employs an imaging spectrometer to acquire depth-resolved profiles from adjacent spatial points without the need for any scanning. To enable this imaging modality, we have considered that signals originating from multiple depths combine in a different manner in FDOCT compared to time-domain optical coherence tomography (TDOCT). Because a multicomponent FDOCT signal is a coherent sum, it is necessary to limit the number of modes that contribute to the detected signal. Conversely, multicomponent TDOCT signals can be represented as incoherent sums, where increasing the number of modes improves the signal.
Current methods for analysis of spectroscopic optical coherence tomography (SOCT) signals suffer from an inherent tradeoff between time (depth) and frequency (wavelength) resolution. Here, we present ...a dual window (DW) method for reconstructing time frequency distributions (TFDs) that applies two orthogonal Gaussian windows that independently determine the spectral and temporal resolution. The effectiveness of the method is demonstrated in simulations and in processing of measured OCT signals that contain fields which vary in time and frequency. The DW method yields TFDs that maintain high spectral and temporal resolution and are free from the artifacts and limitations commonly observed with other processing methods.
Two separate commercial products of kratom Mitragyna speciosa (Korth.) Havil. Rubiaceae were used to generate reference standards of its indole and oxindole alkaloids. While kratom has been studied ...for over a century, the characterization data in the literature for many of the alkaloids are either incomplete or inconsistent with modern standards. As such, full 1H and 13C NMR spectra, along with HRESIMS and ECD data, are reported for alkaloids 1–19. Of these, four new alkaloids (7, 11, 17, and 18) were characterized using 2D NMR data, and the absolute configurations of 7, 17, and 18 were established by comparison of experimental and calculated ECD spectra. The absolute configuration for the N(4)-oxide (11) was established by comparison of NMR and ECD spectra of its reduced product with those for compound 7. In total, 19 alkaloids were characterized, including the indole alkaloid mitragynine (1) and its diastereoisomers speciociliatine (2), speciogynine (3), and mitraciliatine (4); the indole alkaloid paynantheine (5) and its diastereoisomers isopaynantheine (6) and epiallo-isopaynantheine (7); the N(4)-oxides mitragynine-N(4)-oxide (8), speciociliatine-N(4)-oxide (9), isopaynantheine-N(4)-oxide (10), and epiallo-isopaynantheine-N(4)-oxide (11); the 9-hydroxylated oxindole alkaloids speciofoline (12), isorotundifoleine (13), and isospeciofoleine (14); and the 9-unsubstituted oxindoles corynoxine A (15), corynoxine B (16), 3-epirhynchophylline (17), 3-epicorynoxine B (18), and corynoxeine (19). With the ability to analyze the spectroscopic data of all of these compounds concomitantly, a decision tree was developed to differentiate these kratom alkaloids based on a few key chemical shifts in the 1H and/or 13C NMR spectra.
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