Efficient use and recycling of the micronutrient iodide in mammals

Daily ingestion of iodide alone is not adequate to sustain production of the thyroid hormones, tri- and tetraiodothyronine. Proper maintenance of iodide in vivo also requires its active transport into the thyroid and its salvage from mono- and diiodotyrosine that are formed in excess during hormone biosynthesis. The enzyme iodotyrosine deiodinase responsible for this salvage is unusual in its ability to catalyze a reductive dehalogenation reaction dependent on a flavin cofactor, FMN. Initial characterization of this enzyme was limited by its membrane association, difficult purification and poor stability. The deiodinase became amenable to detailed analysis only after identification and heterologous expression of its gene. Site-directed mutagenesis recently demonstrated that cysteine residues are not necessary for enzymatic activity in contrast to precedence set by other reductive dehalogenases. Truncation of the N-terminal membrane anchor of the deiodinase has provided a soluble and stable source of enzyme sufficient for crystallographic studies. The structure of an enzyme·substrate co-crystal has become invaluable for understanding the origins of substrate selectivity and the mutations causing thyroid disease in humans.     

Rapid Determination of Conidial Viability for Entomopathogenic Hyphomycetes Using Fluorescence Microscopy Techniques

The viability of conidia from two species of deuteromycetes fungi pathogenic to insects was determined using two fluorochrome stains, fluorescein diacetate (FDA) and propidium iodide (PI). These stains were used either alone or in combination, and results were compared with standard conidial germination tests. FDA fluoresces bright green in viable conidia and PI fluoresces red in non-viable conidia, when viewed using specific fluorescence microscopic techniques. Conidia from two isolates of Paecilomyces fumosoroseus (Wize) Brown and Smith and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated. Conidia were suspended in deionized water and half of each suspension was treated with microwave radiation to kill all the conidia. Conidia were tested for viability in non-microwaved suspensions in a mixture (ca. 1:1) of viable and non-viable conidial suspensions, and in the microwaved suspensions that contained all non-viable conidia. No significant differences were observed for the four isolates tested between germination tests on water and agar and viability tests conducted with FDA alone or FDA in combination with PI. One isolate of B. bassiana that had been damaged in storage was also tested. Differences were observed between the actual germination and the percentage of viability determined using FDA or FDA plus PI. Damaged conidia maintained a measure of viability and fluoresced green, but did not fully germinate.     

A method for the analysis of six thyroid hormones in thyroid gland by liquid chromatography–tandem mass spectrometry

Perchlorate can competitively inhibit iodide uptake by the thyroid gland (TG) via the sodium/iodide symporter, consequently reducing the production of thyroid hormones (THs). Until recently, the effects of perchlorate on TH homeostasis are being examined through measurement of serum levels of TH, by immunoassay (IA)-based methods. IA methods are fast, but for TH analysis, they are compromised by the lack of adequate specificity. Therefore, selective and sensitive methods for the analysis of THs in TG are needed, for assessment of the effects of perchlorate on TH homeostasis. In this study, we developed a method for the analysis of six THs: l-thyroxine (T₄), 3,3′,5-triiodo-l-thyronine (T₃), 3,3′,5′-triiodo-l-thyronine (rT₃), 3,5-diiodo-l-thyronine (3,5-T₂), 3,3′-diiodo-l-thyronine (3,3′-T₂), and 3-iodo-l-thyronine (3-T₁) in TG, using liquid chromatography (LC)–tandem mass spectrometry (MS/MS). TGs used in this study were from rats that had been placed on either iodide-deficient diet or iodide-sufficient diet, and that had either been provided with perchlorate in drinking water (10 mg/kg/day) or control water. TGs were extracted by pronase digestion and then analyzed by LC–MS/MS. The instrumental calibration range for each TH ranged from 1 to 200 ng/ml and showed a high linearity (r > 0.99). The method quantification limits (LOQs) were determined to be 0.25 ng/mg TG for 3-T₁; 0.33 ng/mg TG for 3,3′- and 3,5-T₂; and 0.52 ng/mg TG for rT₃, T₃, and T₄. Rats were placed on an iodide-deficient or -sufficient diet for 2.5 months, and for the last 2 weeks of that period were provided either perchlorate (10 mg/kg/day) in drinking water or control water. Iodide deficiency and perchlorate administration both reduced TG stores of rT₃, T₃, and T₄. In iodide-deficient rats, perchlorate exacerbated the reduction in levels of THs in TG. With the advances in analytical methodology, the use of LC–MS/MS for measurement of hormone levels in TG will allow more comprehensive evaluations of the hypothalamic-pituitary–thyroid axis.     

Molecular and expression characterization of a nanos1 homologue in Chinese sturgeon, Acipenser sinensis

The nanos gene family was essential for germ line development in diverse organisms. In the present study, the full-length cDNA of a nanos1 homologue in A. sinensis, Asnanos1, was isolated and characterized. The cDNA sequence of Asnanos1 was 1489 base pairs (bp) in length and encoded a peptide of 228 amino acid residues. Multiple sequence alignment showed that the zinc-finger motifs of Nanos1 were highly conserved in vertebrates. By RT-PCR analysis, Asnanos1 mRNAs were ubiquitously detected in all tissues examined except for the fat, including liver, spleen, heart, ovary, kidney, muscle, intestines, pituitary, hypothalamus, telencephalon, midbrain, cerebellum, and medulla oblongata. Moreover, a specific polyclonal antibody was prepared from the in vitro expressed partial AsNanos1 protein. Western blot analysis revealed that the tissue expression pattern of AsNanos1 was not completely coincided with that of its mRNAs, which was not found in fat, muscle and intestines. Additionally, by immunofluoresence localization, it was observed that AsNanos1 protein was in the cytoplasm of primary oocytes and spermatocytes. The presented results indicated that the expression pattern of Asnanos1 was differential conservation and divergence among diverse species.     

The use of trimethylsilyl iodide as a synthon in coordination chemistry

Trimethylsilyl iodide is shown to be an efficient metathetical reagent for preparing transition-metal iodides from the corresponding chlorides, though often complications can cause problems. These include reduction of the starting metal chloride when its oxidation state is high, due to the reaction of iodide, and even oxidation of low-oxidation-state compounds, presumably by incipient silyl cations. Finally, some very inert chlorides, such as of iridium(III), react too slowly with the iodide under the experimental conditions, and simple reaction with solvent becomes predominant.     

Acidic 1,3-propanediaminetetraacetate zinc complexes as a probe of water soluble coordination polymers

In a very acidic solution, potassium 1,3-propanediaminetetraacetate zinc chloride K_2n[ZnCl₂(1,3-H₂pdta)ZnCl₂]_n (1) and its substituted iodide [ZnI₂(H₂O)(1,3-H₄pdta)]_n (2) (H₄pdta = 1,3-propanediaminetetraacetatic acid, C₁₁H₁₈N₂O₈) were isolated. The former was obtained from the reaction of zinc chloride and H₄pdta in pH ∼1.5. Further substitution of 1 results in the formation of iodide 2 with the addition of potassium iodide in acidic solution of pH 0.5. Complex 1 consists of a dimeric anionic unit [ZnCl₂(1,3-H₂pdta)ZnCl₂]²⁻ with strong intra-molecular hydrogen bonds [N1?O2 2.648(4); N1?O4 2.710(4) Å]. In neutral complex 2, an 1,3-pdta ligand links each monomeric unit [Zn(H₂O)I₂] to generate an infinite 1D chain, which extents into a 3D supramolecular structure by very strong inter-molecular hydrogen bonds the [O4?O2b 2.50 (1) Å, bx, y, z + 1]. 1 is soluble in water at room temperature, which is traced by ¹³C NMR experiment.     

Solvent-dependent 2D-coordination polymers of Cu(I) containing a bridging triazolopyrimidine ligand

Two 2D-polymers of Cu(I), [Cu₂(μ-tp)(CH₃CN)(μ-I)(μ₃-I)]_n (1) and [Cu₂(μ-tp)(μ₃-I)₂]_n (2), both obtained from the reaction between copper iodide and 1,2,4-triazolo[1,5-a]pyrimidine (tp) in the presence of KI are described. While in compound 1, tp acts as bridging ligand via N1 and N3 between chair-like [Cu₄I₄] units, in compound 2, tp moieties are placed at both sides of a [Cu_nI_n] kinked layer, displaying also a bridging mode but, in this case, through its atoms N3 and N4. These structural differences between both compounds seem to be due to the solvent of crystallization, being acetonitrile for 1 and water for 2. These polymers are the first examples of Cu(I) compounds based on the nucleobase-analog ligand tp.     

Monitoring Changes in Membrane Polarity,Membrane Integrity,and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC₄(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca²⁺, K⁺, and H⁺, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.     

Characterization of a rapid and reliable method for iodide biomonitoring in serum and urine based on ion chromatography–ICP-mass spectrometry

An appropriate and controlled supply of thyroid hormones is vital for proper body function. In turn, an appropriate synthesis of T3 and T4 in the thyroid gland is dependent on a sufficient and balanced iodide concentration in blood serum. Due to widespread iodine deficiency or some cases of iodine over exposure, iodide biomonitoring in serum is important and it is that biomonitoring approach being closest to the bioavailable I⁻ supply for the thyroid gland. Therefore, this paper describes a biomonitoring method for iodide determination in serum based on ion chromatography–inductively coupled plasma mass spectrometry (IC–ICP-MS). Since in literature only very few data are available on iodide in serum but many in urine the method is also extended to I⁻ monitoring in urine. The method was additionally designed to have short analysis time (8 min) for increased sample throughput, good precision in serial measurement (serum: 4.86%; urine: 1.4%), and day-to-day determination (serum: 5.7%; urine: 2.28%), high accuracy (serum: 105%; urine: 101%) and good recovery (serum: 102%; urine: 99%) even in matrix-rich samples at low I⁻ concentration. Also, investigations were performed to elucidate whether internal standardization during chromatography, sample preparation for protein-matrix removal or matrix-matched calibration are advantageous for analytical performance. Finally, limits of detection (3σ) of 0.12 μg/L or 0.05 μg/L (serum or urine) and limit of quantification (10σ) of 0.39 μg/L or 0.17 μg/L (serum or urine) were achieved.     

A nonradioactive iodide uptake assay for sodium iodide symporter function

The standard assay for sodium iodide symporter (NIS) function is based on the measurement of radioiodide uptake (¹²⁵I) in NIS-expressing cells. However, cost and safety issues have limited the method from being used widely. Here we describe a simple spectrophotometric assay for the determination of iodide accumulation in rat thyroid-derived cells (FRTL5) based on the catalytic effect of iodide on the reduction of yellow cerium(IV) to colorless cerium(III) in the presence of arsenious acid (Sandell-Kolthoff reaction). The assay is fast and highly reproducible with a Z′ factor of 0.70. This procedure allows the screening of more than 800 samples per day and can easily be adapted to robotic systems for high-throughput screening of NIS function modulators. Using this method, the potency of several known inhibitors of NIS function was evaluated in a single day with high accuracy and reliability. Measured IC₅₀ values were essentially identical to those determined using Na¹²⁵I.