Saxitoxin (STX) and its analogues cause the paralytic shellfish poisoning (PSP) syndrome, which afflicts human health and impacts coastal shellfish economies worldwide. PSP toxins are unique alkaloids, being produced by both prokaryotes and eukaryotes. Here we describe a candidate PSP toxin biosynthesis gene cluster (sxt) from Cylindrospermopsis raciborskii T3. The saxitoxin biosynthetic pathway is encoded by more than 35 kb, and comparative sequence analysis assigns 30 catalytic functions to 26 proteins. STX biosynthesis is initiated with arginine, S-adenosylmethionine, and acetate by a new type of polyketide synthase, which can putatively perform a methylation of acetate, and a Claisen condensation reaction between propionate and arginine. Further steps involve enzymes catalyzing three heterocyclizations and various tailoring reactions that result in the numerous isoforms of saxitoxin. In the absence of a gene transfer system in these microorganisms, we have revised the description of the known STX biosynthetic pathway, with in silico functional inferences based on sxt open reading frames combined with liquid chromatography-tandem mass spectrometry analysis of the biosynthetic intermediates. Our results indicate the evolutionary origin for the production of PSP toxins in an ancestral cyanobacterium with genetic contributions from diverse phylogenetic lineages of bacteria and provide a quantum addition to the catalytic collective available for future combinatorial biosyntheses. The distribution of these genes also supports the idea of the involvement of this gene cluster in STX production in various cyanobacteria.
Abstract This study describes the utilization of an LC-MS/MS based H295R assay to assess an environmentally relevant mixture of persistent organic pollutants (POPs). H295R cells were exposed to the POP mixture in two conditions stimulated with 10 μM forskolin and unstimulated. Most importantly, the unstimulated cells responded to the low concentration of the mixture with a significant down-regulation of dehydroepiandrosterone (DHEA). This response was not observed in forskolin-stimulated cells. In stimulated H295R cells, exposure to the highest concentration showed a trend towards induced production of mineralocorticoids and glucocorticoids, although this was not significant. On the other hand, in the same exposure concentration and condition, estrogen and androgen production tended to be down-regulated. In addition to these patterns of responses being different in the stimulated vs unstimulated condition, four steroids were not detectable in the unstimulated condition.
The reference change value (RCV) is calculated by combining the within-subject biological variation (CVI) and local analytical variation (CVA). These calculations do not account for the variation seen in preanalytical conditions in routine practice or CVI in patients presenting for treatment. As a result, the RCVs may not reflect routine practice or align with clinicians' experiences. We propose a novel RCV approach based on routine patient data that is potentially more clinically relevant.
Abstract Background Saxitoxin and its analogues collectively known as the paralytic shellfish toxins (PSTs) are neurotoxic alkaloids and are the cause of the syndrome named paralytic shellfish poisoning. PSTs are produced by a unique biosynthetic pathway, which involves reactions that are rare in microbial metabolic pathways. Nevertheless, distantly related organisms such as dinoflagellates and cyanobacteria appear to produce these toxins using the same pathway. Hypothesised explanations for such an unusual phylogenetic distribution of this shared uncommon metabolic pathway, include a polyphyletic origin, an involvement of symbiotic bacteria, and horizontal gene transfer. Results We describe the identification, annotation and bioinformatic characterisation of the putative paralytic shellfish toxin biosynthesis clusters in an Australian isolate of Anabaena circinalis and an American isolate of Aphanizomenon sp ., both members of the Nostocales . These putative PST gene clusters span approximately 28 kb and contain genes coding for the biosynthesis and export of the toxin. A putative insertion/excision site in the Australian Anabaena circinalis AWQC131C was identified, and the organization and evolution of the gene clusters are discussed. A biosynthetic pathway leading to the formation of saxitoxin and its analogues in these organisms is proposed. Conclusion The PST biosynthesis gene cluster presents a mosaic structure, whereby genes have apparently transposed in segments of varying size, resulting in different gene arrangements in all three sxt clusters sequenced so far. The gene cluster organizational structure and sequence similarity seems to reflect the phylogeny of the producer organisms, indicating that the gene clusters have an ancient origin, or that their lateral transfer was also an ancient event. The knowledge we gain from the characterisation of the PST biosynthesis gene clusters, including the identity and sequence of the genes involved in the biosynthesis, may also afford the identification of these gene clusters in dinoflagellates, the cause of human mortalities and significant financial loss to the tourism and shellfish industries.
The cosyntropin test is used to diagnose adrenal insufficiency (AI) and nonclassical congenital adrenal hyperplasia (NCCAH). Current cutoffs for cortisol and 17-hydroxyprogesterone (17-OHP) are derived from nonstandardized immunoassays. Liquid chromatography tandem mass spectrometry (LC-MS/MS) offers direct measurement of steroids, prompting the need to re-establish normal ranges. The goal of this study was to define cutoff values for cortisol and 17-OHP in serum by LC-MS/MS 30 and 60 minutes after intravenous administration of 250 µg tetracosactide acetate to healthy volunteers and to compare the results with LC-MS/MS with routine immunoassays. Cosyntropin testing was performed in healthy subjects (n = 138) and in patients referred for evaluation of adrenocortical function (n = 94). Steroids were assayed by LC-MS/MS and compared with two immunoassays used in routine diagnostics (Immulite and Roche platforms). The cutoff level for cortisol was defined as the 2.5% percentile in healthy subjects not using oral estrogens (n = 121) and for 17-OHP as the 97.5% percentile. Cortisol cutoff levels for LC-MS/MS were 412 and 485 nmol/L at 30 and 60 minutes, respectively. Applying the new cutoffs, 13 of 60 (22%) subjects who had AI according to conventional criteria now had a normal test result. For 17-OHP, the cutoff levels were 8.9 and 9.0 nmol/L at 30 and 60 minutes, respectively. LC-MS/MS provides cutoff levels for cortisol and 17-OHP after cosyntropin stimulation that are lower than those based on immunoassays, possibly because cross-reactivity between steroid intermediates and cortisol is eliminated. This reduces the number of false-positive tests for AI and false-negative tests for NCCAH.
Saxitoxin (STX) and its analogs are voltage‐gated sodium‐channel blockers that cause paralytic shellfish poisoning (PSP) and negatively affect human health and seafood industries worldwide. Little is known about the molecular biology of PSP‐toxin synthesis. Saxitoxin precursors were identified 25 years ago, and a hypothetical biosynthesis pathway was proposed; however, the correct sequence of reactions and enzymes involved in their catalysis remains to be identified. This study describes the optimization of in vitro biosynthesis of PSP toxins by cellular lysates of the toxic cyanobacterium Cylindrospermopsis raciborskii (Wołosz.) Seenaya et Subbaraju T3 and the characterization of its biochemical requirements. Enzymes involved in PSP‐toxin synthesis are located in the cytosol. The molecular components of in vitro biosynthesis reactions could not be completely defined because of the requirement of an unknown cofactor. Evidence is presented that supports the previous suggestion that STX biosynthesis involves a Claisen condensation between arginine and acetate. In addition, carbamoyl phosphate was identified as a likely precursor for carbamated PSP toxins. Predictions have been made regarding the enzymes that may be involved in the biosynthesis of PSP toxins. These included class II aminotransferase; nonheme iron oxygenase, containing flavin, and possibly ferredoxin, as the prosthetic groups; and an O ‐carbamoyltransferase. On the other hand, the involvement of cytochrome P450 monooxygenase was excluded.
Saxitoxins are neurotoxic alkaloids produced by cyanobacteria and dinoflagellates. Due to their antagonism of voltage-gated sodium channels, they are desirable candidates for drug development as anaesthetics and muscle relaxants. We describe the heterologous expression and biochemical characterization of the first enzyme in the saxitoxin pathway, SxtA, in E. coli. This unusual polyketide synthase-like enzyme is able to condense multiple acid-CoA substrates with arginine and could potentially be exploited for the production of novel compounds of biomedical value. More information can be found in the communication by R. Kellmann, B. Neilan et al. Credits: Bloom photo courtesy of Dr. Nick Crosbie, Melbourne Water. Syringe image by Jupiter Images (sourced through Canva Pro).
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