ABSTRACT
An unusual trithioorthoformate-capped cyclophane cage was assembled via antimony-activated iodine oxidation of thiols as confirmed by 1 H-NMR spectroscopy and X-ray crystallography. The disulfide bridges can undergo desulfurization with hexamethylphosphorous triamide (HMPT) at ambient temperature to capture a trithioether cyclophane cage capped by the trithioorthoformate. In both cages a methine proton points directly into the small cavity. This unexpected structure is hypothesized to have formed as a result of haloform insertion during oxidation.
ABSTRACT
The kisspeptin system plays pivotal roles in the regulation of vertebrate reproduction. Classically, kisspeptin produced in the brain stimulates brain gonadotropin-releasing hormone signaling, which in turn activates the pituitary-gonad axis. Expression of the kisspeptin system has also been documented in peripheral tissues, including gonads of mammals and fishes. However, the fish gonadal kisspeptin system remained uncharacterized. Herein we report identification and characterization of four kisspeptin system mRNAs (kisspeptin 1 (kiss1), kiss2, and G protein-coupled receptor 54-1 (gpr54-1) and gpr54-2) in sablefish, Anoplopoma fimbria. Sablefish predicted protein sequences were highly similar to those of other marine teleosts, but less so to freshwater teleosts. Tissue distribution analyses revealed that all four kisspeptin-system transcripts were expressed in both brain and gonad. However, kiss2 was the predominant transcript in the gonads and the only transcript detected in ovulated eggs. Ontogenetic analysis of kiss2 expression in juvenile sablefish gonads demonstrated that levels were low during sex differentiation but increased with fish size and gonadal development. Dramatic increases in kiss2 mRNA occurred during primary oocyte growth, while levels remained relatively low in testes. In situ hybridization revealed that kiss2 mRNA was localized to cytoplasm of perinucleolus stage oocytes, suggesting it could play a local role in oogenesis or could be synthesized and stored within oocytes as maternal mRNA. This represents the first study to focus on the gonadal kisspeptin system in fishes and provides important tools for further investigation of both the gonadal and brain kisspeptin systems in sablefish.
Subject(s)
Gonads/metabolism , Kisspeptins/metabolism , Perciformes/metabolism , Receptors, G-Protein-Coupled/metabolism , Sex Differentiation/physiology , Amino Acid Sequence , Animals , Brain/metabolism , Cloning, Molecular , Kisspeptins/genetics , RNA, Messenger/genetics , Receptors, G-Protein-Coupled/genetics , ReproductionABSTRACT
Regardless of how sex is determined, the gonadal genes expressed downstream that regulate sex differentiation are relatively conserved among vertebrates. The goal of this study was to clone and characterize five key sex differentiation-related genes in a Scorpaeniform fish, sablefish (Anoplopoma fimbria). Complete mRNA sequences of foxl2, cyp19a1a, dmrt1, sox9a and amh were cloned, sequenced, and phylogenetically analyzed. The sablefish mRNA sequences exhibited the characteristic domains of each gene. The deduced amino sequences were highly conserved in some cases, such as Foxl2, whereas others, such as Amh, exhibited lower homology to corresponding sequences in other vertebrates. Using quantitative PCRs developed for each gene, we found that foxl2 and cyp19a1a mRNA levels were significantly elevated in juvenile sablefish ovaries compared to testes, whereas dmrt1, sox9a and amh mRNA levels were significantly elevated in testes relative to ovaries. These patterns were upheld in our tissue distribution analyses of adult fish, but overall four of the genes, foxl2, cyp19a1a, dmrt1 and amh, were robust markers of sex in sablefish. This study provides important molecular tools for ongoing work related to sex control in sablefish and exploration of the earliest period of molecular sex differentiation and its regulation.
Subject(s)
Fish Proteins/genetics , Fishes/genetics , Sex Differentiation/genetics , Amino Acid Sequence , Animals , Anti-Mullerian Hormone/genetics , Cloning, Molecular , Forkhead Transcription Factors/genetics , Molecular Sequence Data , RNA, Messenger/genetics , SOX9 Transcription Factor/genetics , Sequence Alignment , Steroidogenic Factor 1/genetics , Transcription Factors/geneticsABSTRACT
Immunoassays are commonly used to screen samples prior to confirmation by gas chromatography-mass spectrometry (GC-MS). This serves two purposes: it provides a second method for positive samples, and it allows exclusion of negative samples from further confirmatory testing. In addition, immunoassay results can be used in some cases to determine if dilution of the sample will be required during the confirmatory assay. We used 878 sweat patches worn by 38 subjects receiving treatment for cocaine dependence to compare analysis of the extracts of the patches for cocaine immuno-equivalents by radioimmunoassay (RIA) with determination of cocaine, benzoylecgonine (BE), and ecgonine methyl ester (EME) by GC-MS. Preliminary validation experiments demonstrate that the GC-MS method using positive ion chemical ionization had sufficient specificity and recovery to support a lower limit of quantitation (LLOQ) of 4 ng/patch and was precise and accurate across a linear range up to 500 ng/patch. Cocaine ranging from the LLOQ to 31,900 ng/patch was found in 660 of the samples; BE ranging from the LLOQ to 3470 ng/patch was found in 530 of the samples; and EME ranging from the LLOQ to 2280 ng/patch was found in 476 of the samples. In a subset of 238 samples semiquantitative use of the RIA gave results that agreed with GC-MS with a correlation coefficient of 0.986, but averaged approximately 23% lower. Although this accuracy of the RIA supported its use as a sole quantitative assay, the limited linear range of the RIA (4-200 ng/patch) proved impractical for this purpose. Receiver operator characteristic analysis of the cutoffs of the RIA and GC-MS suggested optimal cutoffs of 5 and 4 ng/patch, respectively. At these cutoffs, the RIA had sensitivity of 90.0% and specificity of 92.2%. For samples that had RIA results greater than the high calibrator (N = 228), various dilution schemes were assessed for their ability to predict retention of either cocaine alone or cocaine and both metabolites within the linear range of the GC-MS. When cocaine was the only analyte of interest, a single 20-fold dilution retained 200 (87.7%) of the samples. This compared to an optimal scheme where a different dilution was selected for each one-tenth ratio (< 0.1, 0.1-0.2, etc.) where 211 (92.5%) of the samples were retained. When trying to retain cocaine and both metabolites, dilution schemes were less successful as BE and EME would often fall below the LLOQ of the GC-MS. A single fivefold dilution of all samples retained 115 (50.4%) compared to an optimum of 143 (62.7%). The optimum could be approached with four dilution sets retaining 142 of the samples. Time expended on performing RIA analysis of all the samples was cost-effective when the results were used to exclude negatives from and predict dilutions required for GC-MS analysis. RIA offers a cost-effective, sensitive, and specific alternative initial test for cocaine determination in extracts of sweat patches.