Influence of mitochondrial inhibitors on the respiration and energy-dependent uptake of iodide by thyroid slices

The influence of mitochondrial inhibitors, including oligomycin, antimycin and rotenone, on the iodide and oxygen uptake and the nucleotide content of incubated sheep thyroid slices was investigated. Each inhibitor strongly suppressed both iodide and oxygen uptake, and decreased the nucleoside triphosphate content of the slices. In most cases the addition of glucose or mitochondrial substrates restored iodide uptake in inhibitor-treated slices. Inhibitor concentrations sufficient to inhibit iodide uptake strongly had only slight effects on the thyroidal Na(+)+K(+)-activated adenosine triphosphatase. It is concluded that the inhibitors produce their effects by the inhibition in vivo of mitochondrial oxidative phosphorylation. ATP synthesis appears to be essential for iodide uptake to occur, and the high-energy intermediates (or energized state) of oxidative phosphorylation cannot be used to energize the uptake process. To a limited extent glycolytic ATP synthesis can support iodide uptake, which is therefore not exclusively dependent on aerobic metabolism. The mechanism of energy-linked iodide uptake is discussed.     

Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability

This study was designed to assess whether nucleic acid stains such as propidium iodide and Hoechst 33258 and the cytosolic stain eosin identified equivalent proportions of non-viable cells. Sub-samples of boar spermatozoa stored for up to 72 h, and frozen bull spermatozoa stored in straws and thawed before staining, were exposed to either propidium iodide or Hoechst 33258 alone or in combination. Additional sub-samples were stained with eosin-nigrosin and subsequently with Giemsa. The proportion of non-viable cells identified by propidium iodide alone was equivalent to that observed when it was used in combination with the other fluorescent probe. Similar results were observed for Hoechst 33258. However, direct microscopic examination of sub-samples exposed to both stains revealed that a proportion of spermatozoa stained with propidium iodide did not incorporate Hoechst 33258. This was found consistently in boar and bull spermatozoa under the different experimental conditions used. Quantification showed that the proportion of propidium iodide-positive cells was significantly higher than Hoechst 33258-positive cells. Furthermore, the proportion of propidium iodide-positive cells was higher than cells stained with eosin, but no differences were found between the number of cells stained with Hoechst 33258 or eosin. The proportion of cells stained with propidium iodide was positively correlated with the proportion stained with either Hoechst 33258 or eosin, despite the observation that more cells incorporated propidium iodide. Taken together, these results indicate that there are differences in the ability of fluorescent probes to identify non-viable sperm cells and that this should be considered when staining protocols are used to analyse sperm viability, or when viability is used as a discriminating factor in functional studies, such as those related to acrosomal exocytosis.     

Effect of triiodothyronine or iodide on the thyroidal secretion in vitro: Inhibition of TSH- and dibutyryl-cyclic-AMP induced endocytosis.

T3 or iodide, at the concentration of 10- minus 3M, inhibits TSH- or dibutyryl-cyclic AMP-induced thyroidal intracellular colloid droplet formation, while leaving the TSH-induced increase in cyclic AMP levels intact. The effect of T3 appeared not to be mediated through the iodide derived from deiodination of T3. Those results clearly demonstrate that T3 or iodide acts at the point beyond cyclic AMP generation and prior tothe formation of intracellular colloid droplets. This is also the site at which microtubule active reagents exert their influence. Whether T3 or iodide fits this category or acts in as yet undetermined manner remains to be established.     

Propidium iodide as an indicator of Giardia cyst viability.

The use of propidium iodide, whose uptake indicates cell death or damage, was investigated to assess the viability of heat-inactivated and chemically inactivated Giardia muris cysts. This was done by comparing propidium iodide staining with excystation. We first determined that propidium iodide could be used with an immunofluorescence detection procedure by showing that the percentages of Giardia lamblia cysts stained with this dye before and after subjecting them to a fluorescence detection method were similar. G. muris cysts were then exposed to heat (56 degrees C), 0.5 to 4 mg of chlorine per liter (pH 7.0, 5 degrees C), 0.1 to 10 mg of a quaternary ammonium compound per liter, or 2 mg of preformed and forming monochloramine per liter (pH 7.2, 18 to 20 degrees C). A good positive correlation between percent propidium iodide-stained cysts and lack of excystation was demonstrated for G. muris cysts exposed either to heat or to the quaternary ammonium compound. However, no significant correlation between absence of excystation and propidium iodide staining was found for cysts exposed to chlorine or monochloramines. These results demonstrate that the propidium iodide staining procedure is not satisfactory for determining the viability of G. muris cysts exposed to these two commonly used drinking water disinfectants.     

Propidium iodide as an indicator of Giardia cyst viability.

The use of propidium iodide, whose uptake indicates cell death or damage, was investigated to assess the viability of heat-inactivated and chemically inactivated Giardia muris cysts. This was done by comparing propidium iodide staining with excystation. We first determined that propidium iodide could be used with an immunofluorescence detection procedure by showing that the percentages of Giardia lamblia cysts stained with this dye before and after subjecting them to a fluorescence detection method were similar. G. muris cysts were then exposed to heat (56 degrees C), 0.5 to 4 mg of chlorine per liter (pH 7.0, 5 degrees C), 0.1 to 10 mg of a quaternary ammonium compound per liter, or 2 mg of preformed and forming monochloramine per liter (pH 7.2, 18 to 20 degrees C). A good positive correlation between percent propidium iodide-stained cysts and lack of excystation was demonstrated for G. muris cysts exposed either to heat or to the quaternary ammonium compound. However, no significant correlation between absence of excystation and propidium iodide staining was found for cysts exposed to chlorine or monochloramines. These results demonstrate that the propidium iodide staining procedure is not satisfactory for determining the viability of G. muris cysts exposed to these two commonly used drinking water disinfectants.     

酵母羧甲基化及光照甘油醛-3-磷酸脱氢酶在碘化钾溶液中荧光发色团的形成

CM-GAPDH在碘化钾溶液中,NAD~+的存在下,形成发射波长为383nm的荧光物。对照的NAD~+与碘化钾溶液混合不产生荧光物。全位及半位修饰光照酶的内源荧光在碘化钾溶液中的变化与天然酶的有明显不同。两者在碘化钾中都形成383nm的荧光,但全位修饰光照酶形成383nm荧光的最适碘化钾浓度为1.0M;半位修饰的为0.8M。以上结果暗示:383nm荧光物的形成需要GAPDH和NAD~+同时存在,并且与活性部位巯基修饰的多少有关,该荧光物可能位于GAPDH的活性部位。     

酵母羧甲基化及光照甘油醛-3-磷酸脱氢酶在碘化钾溶液中荧光发色团的形成

CM-GAPDH在碘化钾溶液中,NAD~+的存在下,形成发射波长为383nm的荧光物。对照的NAD~+与碘化钾溶液混合不产生荧光物。全位及半位修饰光照酶的内源荧光在碘化钾溶液中的变化与天然酶的有明显不同。两者在碘化钾中都形成383nm的荧光,但全位修饰光照酶形成383nm荧光的最适碘化钾浓度为1.0M;半位修饰的为0.8M。以上结果暗示:383nm荧光物的形成需要GAPDH和NAD~+同时存在,并且与活性部位巯基修饰的多少有关,该荧光物可能位于GAPDH的活性部位。     

The mammary gland iodide transporter is expressed during lactation and in breast cancer

The sodium/iodide symporter mediates active iodide transport in both healthy and cancerous thyroid tissue. By exploiting this activity, radioiodide has been used for decades with considerable success in the detection and treatment of thyroid cancer. Here we show that a specialized form of the sodium/iodide symporter in the mammary gland mediates active iodide transport in healthy lactating (but not in nonlactating) mammary gland and in mammary tumors. In addition to characterizing the hormonal regulation of the mammary gland sodium/iodide symporter, we demonstrate by scintigraphy that mammary adenocarcinomas in transgenic mice bearing Ras or Neu oncogenes actively accumulate iodide by this symporter in vivo. Moreover, more than 80% of the human breast cancer samples we analyzed by immunohistochemistry expressed the symporter, compared with none of the normal (nonlactating) samples from reductive mammoplasties. These results indicate that the mammary gland sodium/iodide symporter may be an essential breast cancer marker and that radioiodide should be studied as a possible option in the diagnosis and treatment of breast cancer.     

Comparative Study of Microsporidian Spores by Flow Cytometric Analysis

ABSTRACT. Spore suspensions of microsporidian parasites of fish (Microsporidium ovoideum, Glugea stephani, Glugea atherinae and Spraguea lophii ) have been analyzed by flow cytometry. Spore nuclei were dyed either by propidium iodide or bis-benzimide (Hoechst 33342). By observation of forward light scatter and fluorescence the four species could be distinguished and the mono- and diplokaryotic populations of S. lophii identified. Staining of DNA by bis-benzimide was better and easier than propidium iodide. Forward light scatter and fluorescence values were characteristic of each species and remained unchanged throughout the year, so flow cytometry can be used for distinction of spores of some microsporidian parasites once their flow cytometric parameters are known. However, special care has to be taken in tool calibration and material preparation for analysis because of the high precision of the technique.     

Relationship of iodide-induced increase in TSH response to TRH to changes in serum thyroid hormones.

Large doses of iodide (500 mg three times a day) administered to normal men for 10--12 days caused a rise in basal serum TSH and a concomitant rise in the peak TSH response to TRH. The basal and peak levels of TSH were highly correlated (p less than 0.001). However, the iodide-induced rise in the peak TSH after TRH was poorly correlated with concomitant changes in serum thyroid hormones. Serum T3 wa not lower after iodide and, while serum T4 was somewhat lower, the fall in serum T4 was unexpectedly inversely rather than directly correlated with the rise in the peak TSH response to TRH. Thus, increased TSH secretion after iodide need not always be directly correlated with decreased concentrations of circulating thyroid hormones even when large doses of iodide are used. Clinically, a patient taking iodide may have an increased TSH response in a TRH stimulation test even though there is little or no change in the serum level of T3 or T4.     

Direct evidence for ATP consumption due to iodide uptake by isolated, uncultured, pig thyroid epithelial cells

The concept that iodide uptake by thyroid requires a supply of ATP is currently accepted. However, there is little direct evidence that the extents of iodide uptake and ATP consumption are correlated. To demonstrate this correlation, we used isolated, uncultured pig thyroid epithelial cells in basal media containing only glucose and the cations necessary for Na+,K(+)-ATPase, which prevented other possible cellular activities requiring ATP. The isolated cells were sensitive to various metabolic inhibitors of ATP-generating systems, confirming that they were intact. The extents of increase in iodide uptake and decrease in ATP content of the cells were entirely dependent on the presence of the cations, and were closely correlated with each other. Furthermore, the ATP content was not reduced in the absence of NaI, even when all necessary cations for the ATPase were present. These findings provide direct evidence for a correlation between the extents of iodide uptake and ATP consumption. Cells in medium with Mg alone did not show iodide incorporation or ATP consumption, confirming that Mg2(+)-dependent ATPase does not contribute to iodide uptake by the thyroid.     

Effect of anions on the cation selectivity of gramicidin-containing liposomes

Summary An osmotic method was used to study the salt permeability induced by gramicidin A in liposomes. Sequences of cation permeation were obtained for iodide, salycilate, acetate und formate salts in liposomes below and above their transition temperature. Salycilate and formate salts, unlike acetate and iodide salts, exhibit the same sequences for cation selectivity in liposomes below and above their transition temperature. These results can be explained by assuming three mechanisms for salt permeation across gramicidin-containing liposomes: (i) the anion moves by the lipid part of the membrane whereas the cation moves by the gramicidin channel, (ii) movement of the undissociated acid species occurs through the lipid part of the membrane followed by cation-proton exchange via the gramicidin channel and (iii) the cation and anion may move simultaneously via the gramicidin channel.When the movement of the anion or undissociated acid across the lipid part of the membrane is not rate limiting the permeation process, the cation selectivity obtained agrees with the cation selectivity of the gramicidin A channel, as determined by others using independent measurements.     

Effect of anions on the cation selectivity of gramicidin-containing liposomes

An osmotic method was used to study the salt permeability induced by gramicidin A in liposomes. Sequences of cation permeation were obtained for iodide, salycilate, acetate and formate salts in liposomes below and above their transition temperature. Salycilate and formate salts, unlike acetate and iodide salts, exhibit the same sequences for cation selectivity in liposomes below and above their transition temperature. These results can be explained by assuming three mechanisms for salt permeation across gramicidin-containing liposomes: (i) the anion moves by the lipid part of the membrane whereas the cation moves by the gramicidin channel, (ii) movement of the undissociated acid species occurs through the lipid part of the membrane followed by cation-proton exchange via the gramicidin channel and (iii) the cation and anion may move simultaneously via the gramicidin channel. When the movement of the anion or undissociated acid across the lipid part of the membrane is not rate limiting the permeation process, the cation selectivity obtained agrees with the cation selectivity of the gramicidin A channel, as determined by others using independent measurements.     

Classification of larval circulating hemocytes of the silkworm,<Emphasis Type="Italic"> Bombyx mori</Emphasis>, by acridine orange and propidium iodide staining

Circulating hemocytes of the silkworm can be classified by fluorescence microscopy following staining with acridine orange and propidium iodide. Based on their fluorescence characteristics, three groups of circulating hemocytes can be distinguished. The first group, granulocytes and spherulocytes, is positive for acridine orange and contain bright green fluorescent granules when observed by fluorescence microscopy. In granulocytes, these green granules are heterogeneous and relatively small. In contrast, in spherulocytes, the green granules appear more homogenous and larger. The second group of hemocytes consists of prohemocytes and plasmatocytes. These cells appear faint green following staining with acridine orange and do not contain any green fluorescent granules in the cytoplasm. Prohemocytes are round, and their nuclei are dark and clear within a background of faint green fluorescence. Inside the nucleus there are one or two small bright green fluorescent bodies. Plasmatocytes are irregularly shaped and their nuclei are invisible. Oenocytoids belong to the third group, and their nuclei are positive for propidium iodide. Therefore, all five types of circulating hemocytes of the silkworm, including many peculiar ones that are difficult to identify by light microscopy, can now be easily classified by fluorescence microscopy following staining with acridine orange and propidium iodide. In addition, we show that hemocytes positive for acridine orange and propidium iodide are in fact living cells based on assays for hemocyte composition, phagocytosis, and mitochondrial enzyme activity.     

Flow cytometric quantification of electroporation-mediated uptake of macromolecules into plant protoplasts

Summary Flow cytometry was used to provide a rapid and accurate assessment of electroporation-induced uptake of macromolecules into plant protoplasts. Rice protoplasts were electroporated in the presence of fluorescein isothiocyanate-conjugated dextran (FITC-dextran). After washing, the protoplasts were resuspended in a solution containing propidium iodide which intercalates with DNA, but which is excluded by an intact plasma membrane. Electroporation in the presence of FITC-dextran gave rise to populations of protoplasts that fluoresced green or yellow due to the presence of non-conjugated FITC. Non-viable protoplasts fluoresced red because of their inability to exclude propidium iodide molecules. Flow cytometry was used to resolve and quantify these protoplast populations and thus identify optimal conditions for macromolecule uptake. A direct relationship was observed between FITC-dextran uptake and transient gene expression following plasmid uptake. Thus, simultaneous electroporation of protoplasts with foreign DNA and FITC-dextran followed by fluorescence activated cell sorting may permit partial selection of transformed cells and so reduce the need for a selectable marker.Abbreviations ADC analogue to digital converter - CAT chloramphenicol acetyl transferase (enzyme) - cat chloramphenicol acetyl transferase (gene) - CPW solution cell and protoplast wash solution - DC direct current - EF electrofusion - FALS forward angle light scatter - FITC fluorescein isothiocyanate - FITC-dextran fluorescein isothiocyanate conjugated dextran - PI propidium iodide - PMT photomultipliertube - TLC thin layer chromatography     

Synthesis of 3H and 13C labeled mRNA cap dinucleotides--useful tools for NMR, biochemical, and biological studies

For deeper understanding the roles of the mRNA cap structure in cellular processes isotopically labeled dinucleotide cap analogues have been synthesized as tools for NMR and in vivo studies. Tritium or carbon C-13 labeled methyl iodide was used as a source of the isotope material. In order to minimize the number of steps during the radioisotopic synthesis the methylation with tritium labeled methyl iodide was performed with Gp(3)G as a substrate. The C-13 isotope was introduced into the cap dinucleotide by methylation of GDP with C-13 Methyl iodide, followed by coupling the product with guanosine 5'-phosphorimidazolide in DMF with zinc chloride as a catalyst.     

Role of iodine in antioxidant defence in thyroid and breast disease

The role played in thyroid hormonogenesis by iodide oxidation to iodine (organification) is well established. Iodine deficiency may produce conditions of oxidative stress with high TSH producing a level of H_2O_2, which because of lack of iodide is not being used to form thyroid hormones. The cytotoxic actions of excess iodide in thyroid cells may depend on the formation of free radicals and can be attributed to both necrotic and apoptotic mechanisms with necrosis predominating in goiter development and apoptosis during iodide induced involution. These cytotoxic effects appear to depend on the status of antioxidative enzymes and may only be evident in conditions of selenium deficiency where the activity of selenium containing antioxidative enzymes is impaired. Less compelling evidence exists of a role for iodide as an antioxidant in the breast. However the Japanese experience may indicate a protective effect against breast cancer for an iodine rich seaweed containing diet. Similarly thyroid autoimmunity may also be associated with improved prognosis. Whether this phenomenon is breast specific and its possible relationship to iodine or selenium status awaits resolution.     

Metabolism of 131I in relation to strobilation of Aurelia aurita L. (Scyphozoa)

The ¹³¹I isotope of iodine has been used to follow the uptake and metabolism of iodine during the process of strobilation in pre-conditioned polyps of Aurelia aurita L. Strobilating polyps accumulated free iodide from the media against a concentration gradient, the segmented portion of the polyps accumulating about three times as much as the basal portion. Almost all of the accumulated iodide appeared in the soluble portion of an acid-ethanol extract of polyp tissue as inorganic iodide. The time course of accumulation of iodine was not affected by previous exposure to either higher temperature or iodide. Inorganic iodide rather than organically bound iodine is thought to be the effective factor in the initiation of strobilation.     

Alteration in tissue and serum concentrations of TSH, iodide, T4 and T3 induced by various dietary iodide levels.

Isotopic equilibrium and radioimmunoassay methods were used to evaluate the effects of increases in iodide intake on tissue and serum concentrations of thyroid hormones. Within the range of iodide levels used total iodine in peripheral tissues and serum increase directly with iodide intake but this change is mainly due to an increase in inorganic iodide. It is concluded that increases in tissue thyroid hormone concentrations occur within a relatively narrow range of iodide intake and maximal concentration occurs at an iodide intake of 3-10 mug/day.     

Intracellular and extracellular sites of iodination in dispersed hog thyroid cells.

Iodination and hormone synthesis has been studied in isolated hog thyroid cells in suspension. We characterized three iodination processes by use of pharmacological agents. (1) Intracellular iodination dependent on active iodide transport, which was inhibited by NaClO4 or ouabain, but not by catalase. This iodination was linear for 6h with no apparent Km for iodide of 1.5 muM, was stimulated by thyrotropin or N6O2'-dibutyryladenosine 3':5'-cyclic monophosphate, yielded mostly iodinated thyroglobulin and was efficient for tetraiodothyronine synthesis. (2) Extracellular iodination, which was sensitive to catalase, but not to NaClO4 or ouabain. This iodination plateaued after 2h and the apparent Km was 16.5 muM. This process was insensitive to thyrotropin and dibutyryl cyclic AMP. The major products were iodoprotein other then thyroglobulin and iodolipid and the yield of tetraiodothyronine was low. (3) Intracellular iodination from passively diffused iodide, which was not sensitive to inhibitors. Other characteristics of passive intracellular iodination were intermediate between active intracellular iodination and extracellular iodination. The fact that the three processes are inhibited by similar concentrations of methimazole, and their apparent Km values, when corrected for the concentrating effect of iodide trapping, are all of the same order as the Km of purified thyroid peroxidases, suggest that although their locations are different, the enzymic systems involved are identical. These results show that, besides an extracellular site of iodination, dispersed thyroid cells process an intracellular site of iodination with biochemical characteristics of physiological relevance.