Interaction of bis(ethylene)tin(bis(salicylidene)ethylenediamine) with DNA.
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The fluorescence spectral characteristics and interaction of bis(ethylene)tin(bis(salicylidene)ethylenediamine) [Et2Sn(salen)] with DNA are described. The polarity of the solvent has a strong effect on the fluorescence characteristics of Et2Sn(salen). Et2Sn(salen) bound to DNA showed a marked decrease in the fluorescence intensity with a bathochromic shift of the excitation and emission peaks. A hypochromism in the UV absorption spectra was also observed. KI quenching and competitive binding to DNA between Et2Sn(salen) and ethidium bromide (EB) were studied in connection with other experimental observations to show that the interactive model between Et2Sn(salen) and DNA is an intercalative one. The pH and salt effect on the fluorescence properties was also investigated. The intrinsic binding constant was estimated to be 1.071 x 10(5) mol L(-1) in base pairs and the binding site number is 1.98, respectively. A linear relationship between F/F0 and the concentration of calf thymus DNA covers 5.1 x 10(-6) - 2.41 x 10(-4) mol L(-1), which can be utilized for determining traces of calf thymus DNA with a detection limit of 1.1 x 10(-7) mol L(-1) in base pairs. (+info)
Interactions between PEG and type I soluble tumor necrosis factor receptor: modulation by pH and by PEGylation at the N terminus.
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The effects of polyethylene glycol (PEG) on protein structure and the molecular details that regulate its association to polypeptides are largely unknown. These issues were addressed using type I soluble tumor necrosis factor receptor (sTNF-RI) as a model system. Changes in solution viscosity established that a truncated form of sTNF-RI bound free PEG in a pH-dependent manner. Above pH 5.3, the viscosity escalated as the pH increased, while no effect occurred below pH 5.0. Conjugation of 2 kD, 5 kD, or 20 kD PEG to the N terminus attenuated the viscosity at the higher pH values. Tryptophan phosphorescence spectroscopy correlated changes in the protein structure about Trp-107, at the C terminus, with the pH-dependent and PEGylation-dependent attenuation of the viscosity. The results indicate that specific interactions between PEG and the truncated form of sTNF-RI are elicited by an increased flexibility of the truncated protein combined perhaps with removal of steric or charge barriers. Covalently bound PEG at the N terminus reduced the protein affinity for the free polymer and induced a more rigid and polar configuration around Trp-107. Deprotonation of His-105, which is perpendicular to Trp-107, was integral to the binding mechanism producing a pH-dependent switching mechanism. These findings stress the importance of surface charge and structural plasticity in determining macromolecular binding affinities and demonstrate the ability of conjugated PEG to modify the localized surface structure in proteins away from the site of conjugation. (+info)
The effect of iodide administration on hog thyroid gland and the composition of thyroglobulin and 27-S iodoprotein.
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The effect of excess iodide on hog thyroid gland has been examined with regard to the change in the chemical composition of thyroglobulin and in the accumulation of 27-S iodoprotein by the in vivo treatment of hogs with iodide for various lengths of time. The iodine content of thyroglobulin was either unchanged by short term administration of excess iodide, or somewhat lowered. However, the iodine content as well as the total amount of thyroglobulin increased in the glands enlarged by prolonged treatment with iodide. The iodine highest reached 1.17% of the protein on an average. On the other hand, 27-S iodoprotein decreased and finally disappeared after the chronic treatment. Monoiodotyrosine and diiodotyrosine increased in parallel with the increase in the iodine content (0.15 to 1.17%) caused by the iodide treatment, while thyroxine increased but reached a plateau at the level of three residues per mole of thyroglobulin, and no change was observed even in the proteins with the higher iodine content than 0.75%. Proteolytic activity measured by amino acid release from the thyroid protein was depressed by the chronic treatment. On the other hand, the amount of iodocompound released by the autoproteolysis, which may reflect hormone secretion, increased, possibly because of the marked increase in the iodine content of thyroglobulin. (+info)
Reactive oxygen species in the elongation zone of maize leaves are necessary for leaf extension.
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The production and role of reactive oxygen species (ROS) in the expanding zone of maize (Zea mays) leaf blades were investigated. ROS release along the leaf blade was evaluated by embedding intact seedlings in 2',7'-dichlorofluorescein-containing agar and examining the distribution of 2',7'-dichlorofluorescein fluorescence along leaf 4, which was exposed by removing the outer leaves before embedding the seedling. Fluorescence was high in the expanding region, becoming practically non-detectable beyond 65 mm from the ligule, indicating high ROS production in the expansion zone. Segments obtained from the elongation zone of leaf 4 were used to assess the role of ROS in leaf elongation. The distribution of cerium perhydroxide deposits in electron micrographs indicated hydrogen peroxide (H(2)O(2)) presence in the apoplast. 2',7'-Dichlorofluorescein fluorescence and apoplastic H(2)O(2) accumulation were inhibited with diphenyleneiodonium (DPI), which also inhibited O*(2)(-) generation, suggesting a flavin-containing enzyme activity such as NADPH oxidase was involved in ROS production. Segments from the elongation zone incubated in water grew 8% in 2 h. KI treatments, which scavenged H(2)O(2) but did not inhibit O*(2)(-) production, did not modify growth. DPI significantly inhibited segment elongation, and the addition of H(2)O(2) (50 or 500 microM) to the incubation medium partially reverted the inhibition caused by DPI. These results indicate that a certain concentration of H(2)O(2) is necessary for leaf elongation, but it could not be distinguished whether H(2)O(2), or other ROS, are the actual active agents. (+info)
Effect of iodine or iopanoic acid on thyroid Ca2+/NADPH-dependent H2O2-generating activity and thyroperoxidase in toxic diffuse goiters.
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OBJECTIVE: The aim of the present study was to compare the effects of iopanoic acid (IOP) or a saturated solution of potassium iodide (SSKI) administration to patients with toxic diffuse goiters (TDG). DESIGN: Patients with TDG are treated with thionamides and high doses of iodine preoperatively. In this study, two types of preoperative drug regimens were used: propylthiouracil or methimazole plus SSKI for 10-15 days (n=8) or IOP for 7 days (n=6). METHODS: Serum thyroid hormones (total and free thyroxine (T(4)), total tri-iodothyronine (T(3)) and reverse T(3) (rT(3)), were evaluated after 7 days of either SSKI or IOP treatment, and after 10-15 days of SSKI administration. During thyroidectomy, samples of thyroid gland were obtained to evaluate thyroperoxidase and thyroid H(2)O(2)-generating activities. RESULTS: Serum total T(3) was significantly decreased after 7 days of either treatment, and serum rT(3) was significantly increased in IOP-treated patients. Serum total and free T(4) were unaffected by 7 days of IOP treatment, but decreased after 7 days of SSKI treatment, although significantly diminished levels were only reached after a further 3-8 days of SSKI administration. During both drug regimens, serum TSH remained low (SSKI: 0.159+/-0.122; IOP: 0.400+/-0.109 microU/ml). Thyroperoxidase activity was significantly lower in thyroid samples from patients treated with SSKI for 10-15 days than in the thyroid glands from IOP-treated patients. However, thyroid H(2)O(2) generation was inhibited in samples from patients treated with either IOP or SSKI. CONCLUSIONS: We show herein that IOP treatment can be effective in the management of hyperthyroidism and that this drug inhibits thyroid NADPH oxidase activity, just as previously described for SSKI, probably due to its iodine content. (+info)
Inhibition of human hemoglobin autoxidation by sodium n-dodecyl sulphate.
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The effect of sodium n-dodecyl sulphate (SDS) on hemoglobin autoxidation was studied in the presence of a 100 mM phosphate buffer (pH 7.0) by different methods. These included spectrophotometry, fluorescence technique, cyclic voltametry, differential scanning calorimetry, and densitometry. Spectroscopic studies showed that SDS concentrations up to 1 mM increased deoxy-, decreases oxy-, and had no significant effect on the met- conformation of hemoglobin. Therefore, a SDS concentration up to 1 mM increased the deoxy form of hemoglobin as the folded, compact state and decreases the oxy conformation. The turbidity measurements and differential scanning calorimetry techniques indicated a more stable conformation for hemoglobin in the presence of SDS up to 1 mM. Electrochemical studies also confirmed a more difficult oxidation under these conditions. The induction of the deoxy form in the presence of SDS was confirmed by densitometry techniques. The compact structure of deoxyhemoglobin blocks the formation of met-conformation in low SDS concentrations. (+info)
Starch biosynthesis during pollen maturation is associated with altered patterns of gene expression in maize.
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Starch biosynthesis during pollen maturation is not well understood in terms of genes/proteins and intracellular controls that regulate it in developing pollen. We have studied two specific developmental stages: "early," characterized by the lack of starch, before or during pollen mitosis I; and "late," an actively starch-filling post-pollen mitosis I phase in S-type cytoplasmic male-sterile (S-CMS) and two related male-fertile genotypes. The male-fertile starch-positive, but not the CMS starch-deficient, genotypes showed changes in the expression patterns of a large number of genes during this metabolic transition. In addition to a battery of housekeeping genes of carbohydrate metabolism, we observed changes in hexose transporter, plasma membrane H(+)-ATPase, ZmMADS1, and 14-3-3 proteins. Reduction or deficiency in 14-3-3 protein levels in all three major cellular sites (amyloplasts [starch], mitochondria, and cytosol) in male-sterile relative to male-fertile genotypes are of potential interest because of interorganellar communication in this CMS system. Further, the levels of hexose sugars were significantly reduced in male-sterile as compared with male-fertile tissues, not only at "early" and "late" stages but also at an earlier point during meiosis. Collectively, these data suggest that combined effects of both reduced sugars and their reduced flux in starch biosynthesis along with a strong possibility for altered redox passage may lead to the observed temporal changes in gene expressions, and ultimately pollen sterility. (+info)
In vitro assays demonstrate that pollen tube organelles use kinesin-related motor proteins to move along microtubules.
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The movement of pollen tube organelles relies on cytoskeletal elements. Although the movement of organelles along actin filaments in the pollen tube has been studied widely and is becoming progressively clear, it remains unclear what role microtubules play. Many uncertainties about the role of microtubules in the active transport of pollen tube organelles and/or in the control of this process remain to be resolved. In an effort to determine if organelles are capable of moving along microtubules in the absence of actin, we extracted organelles from tobacco pollen tubes and analyzed their ability to move along in vitro-polymerized microtubules under different experimental conditions. Regardless of their size, the organelles moved at different rates along microtubules in the presence of ATP. Cytochalasin D did not inhibit organelle movement, indicating that actin filaments are not required for organelle transport in our assay. The movement of organelles was cytosol independent, which suggests that soluble factors are not necessary for the organelle movement to occur and that microtubule-based motor proteins are present on the organelle surface. By washing organelles with KI, it was possible to release proteins capable of gliding carboxylated beads along microtubules. Several membrane fractions, which were separated by Suc density gradient centrifugation, showed microtubule-based movement. Proteins were extracted by KI treatment from the most active organelle fraction and then analyzed with an ATP-sensitive microtubule binding assay. Proteins isolated by the selective binding to microtubules were tested for the ability to glide microtubules in the in vitro motility assay, for the presence of microtubule-stimulated ATPase activity, and for cross-reactivity with anti-kinesin antibodies. We identified and characterized a 105-kD organelle-associated motor protein that is functionally, biochemically, and immunologically related to kinesin. This work provides clear evidence that the movement of pollen tube organelles is not just actin based; rather, they show a microtubule-based motion as well. This unexpected finding suggests new insights into the use of pollen tube microtubules, which could be used for short-range transport, as actin filaments are in animal cells. (+info)