The photorelease of nitrogen monoxide (NO) from pentacyanonitrosyl coordination compounds of group 8 metals.

The photodetachment of NO from [M(II)(CN)5NO]2- with M = Fe, Ru, and Os, upon laser excitation at various wavelengths (355, 420, and 480 nm) was followed by various techniques. The three complexes showed a wavelength-dependent quantum yield of NO production Phi(NO), as measured with an NO-sensitive electrode, the highest values corresponding to the larger photon energies. For the same excitation wavelength the decrease of Phi(NO) at 20 degrees C in the order Fe > Ru > Os, is explained by the increasing M-N bond strength and inertness of the heavier metals. Transient absorption data at 420 nm indicate the formation of the [M(III)(CN)5H2O]2- species in less than ca. 1 micros for M = Fe and Ru. The enthalpy content of [Fe(III)(CN)5H2O]2- with respect to the parent [Fe(II)(CN)5NO]2- state is (190 +/- 20) kJ mol(-1), as measured by laser-induced optoacoustic spectroscopy (LIOAS) upon excitation at 480 nm. The production of [Fe(III)(CN)5H2O]2- is concomitant with an expansion of (8 +/- 3) ml mol(-1) consistent with an expansion of the water bound through hydrogen bonds to the CN ligands plus the difference between NO release into the bulk and water entrance into the first coordination sphere. The activated process, as indicated by the relatively strong temperature dependence of the Phi(NO) values and by the temperature dependence of the appearance of the [Fe(III)(CN)5H2O]2- species, as determined by LIOAS, is attributed to NO detachment in less than ca. 100 ns from the isonitrosyl (ON) ligand (MS1 state).     

Nitrogen monoxide (no) and glucose: unexpected links between energy metabolism and no-mediated iron mobilization from cells

Nitrogen monoxide (NO) affects cellular iron metabolism due to its high affinity for this metal ion. Indeed, NO has been shown to increase the mRNA binding activity of the iron-regulatory protein 1, which is a major regulator of iron homeostasis. Recently, we have shown that NO generators increase (59)Fe efflux from cells prelabeled with (59)Fe-transferrin (Wardrop, S. L., Watts, R. N., and Richardson, D. R. (2000) Biochemistry 39, 2748-2758). The mechanism involved in this process remains unknown, and in this investigation we demonstrate that it is potentiated upon adding d-glucose (d-Glc) to the reincubation medium. In d-Glc-free or d-Glc-containing media, 5.6 and 16.5% of cellular (59)Fe was released, respectively, in the presence of S-nitrosoglutathione. This difference in (59)Fe release was observed with a variety of NO generators and cell types and was not due to a change in cell viability. Kinetic studies showed that d-Glc had no effect on the rate of NO production by NO generators. Moreover, only the metabolizable monosaccharides d-Glc and d-mannose could stimulate NO-mediated (59)Fe mobilization, whereas other sugars not easily metabolized by fibroblasts had no effect. Hence, metabolism of the monosaccharides was essential to increase NO-mediated (59)Fe release. Incubation of cells with the citric acid cycle intermediates, citrate and pyruvate, did not enhance NO-mediated (59)Fe release. Significantly, preincubation with the GSH-depleting agents, l-buthionine-[S,R]-sulfoximine or diethyl maleate, prevented NO-mediated (59)Fe mobilization. This effect was reversed by incubating cells with N-acetyl-l-cysteine that reconstitutes GSH. These results indicate that GSH levels are essential for NO-mediated (59)Fe efflux. Hence, d-Glc metabolism via the hexose monophosphate shunt resulting in the generation of GSH may be essential for NO-mediated (59)Fe release. These results have important implications for intracellular signaling by NO and also NO-mediated cytotoxicity of activated macrophages that is due, in part, to iron release from tumor target cells.     

The formation of ferritin from apoferritin. Kinetics and mechanism of iron uptake

Ferritin has a high capacity as an iron store, incorporating some 4500 iron atoms as a microcrystalline ferric oxide hydrate. Starting from apoferritin, or ferritin of low iron content, Fe²⁺ and an oxidizing agent, the uptake of iron can be recorded spectrophotometrically. Progress curves were obtained and the reconstituted ferritin was shown by several physical methods to be similar to natural ferritin. The progress curves of iron uptake by apoferritin are sigmoidal; those for ferritins of low iron content are hyperbolic. The rate of iron uptake is dependent on the amount of iron already present in the molecule. The distribution of iron contents among reconstituted ferritin molecules is inhomogeneous. These findings are interpreted in terms of a crystal growth model. The surface area of the crystallites forming inside the protein increases until the molecule is half full, and then declines. This surface controls the rate at which new material is deposited. The experimental results can best be accounted for by a two-stage mechanism, an initial slow `nucleation' stage, which is apparently zero order with respect to [Fe<sup>2+</sup>], followed by a more rapid `growth' stage. The rate of Fe²⁺ oxidation is increased in the presence of apoferritin as compared with controls. Ferritin can therefore be regarded as an enzyme to which the product remains firmly attached. The protein appears to increase the rate of `nucleation'. The apparent zero order of this stage suggests the presence of binding sites on the protein, which are saturated with respect to Fe²⁺. These sites are presumed also to be oxidation sites. The oxidation and subsequent formation of the ferric oxide hydrate may proceed according to one of three alternative models.     

The role of hyperhomocysteinemia in nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation.

Hyperhomocysteinemia (HHcy) is associated with impaired endothelial-dependent vasodilatation and increased risk of atherosclerosis and thrombosis. Here, we summarize some of our previous work on the effect of HHcy on pathways involved in endothelium-dependent vasodilatation, and present new data concerning the endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation. We showed that the 894 G>T single-nucleotide polymorphism in the human endothelial nitric oxide synthase gene (eNOS) increased the risk of recurrent venous thrombosis in individuals with elevated homocysteine levels, indicating that the pathophysiological mechanism in HHcy involves impaired NO-mediated vasodilatation. In addition, the EDHF-mediated vasodilatation of the renal artery was disturbed in diet-induced hyperhomocysteinemic rats. Interestingly, we demonstrated that pretreatment of rats with periodate-oxidized adenosine (Adox), which is an inhibitor of S-adenosylhomocysteine hydrolase, prevented the methionine-induced rise in plasma total Hcy (tHcy) levels but not the inhibition of the EDHF pathway. Furthermore, we demonstrated that S-adenosylhomocysteine (AdoHcy) and S-adenosylmethionine (AdoMet) levels were increased in the kidneys of diet-induced HHcy rats, resulting in a decreased AdoMet:AdoHcy ratio. In addition, we demonstrated that mRNA expression of Connexin 40, which is one of the structural subunits of gap-junctions, was down-regulated in endothelial cells of HHcy rats, and correlated with elevated AdoHcy levels in kidney of these rats. These finding suggest a key role for AdoHcy in relation to decreased Cx40 mRNA expression and impaired EDHF-mediated vasodilatation of HHcy rats.     

Electron paramagnetic resonance spectroscopy with N-methyl-D-glucamine dithiocarbamate iron complexes distinguishes nitric oxide and nitroxyl anion in a redox-dependent manner: applications in identifying nitrogen monoxide products from nitric oxide synthase

Though a large number of studies indicate that nitric oxide synthase (NOS) is responsible for NO&z.rad; production in biological systems, controversy still remains concerning whether NOS directly produces NO&z.rad;. Schmidt et al. (PNAS 93:144492, 1996) proposed that NOS first synthesizes nitroxyl anion (NO(-)), which is then converted to NO&z.rad; by superoxide dismutase (SOD). With electron paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarbamate iron (Fe-MGD), we directly detected NO&z.rad; from purified NOS in the absence of SOD (Xia et al., PNAS 94:12705, 1997). We also showed that the requirement for SOD in the previous NO&z.rad; measurements appeared to be due to the high levels of exogenous superoxide production in their reaction system because of the presence of free FAD. However, it was recently questioned whether Fe-MGD can discriminate NO&z.rad; from NO(-) (Komarov et al., FRBM 28:739-742, 2000). In this study we examined the trapping specificity of different redox forms of Fe-MGD. With Fe(2+)-MGD, NO&z.rad; generated characteristic triplet NO&z.rad;-Fe(2+)-MGD signals (g = 2. 04, a(N) = 12.7 G), whereas NO(-) from Angeli's salt was EPR silent. Both NO&z.rad; and NO(-) gave rise to NO&z.rad;-Fe(2+)-MGD signals when Fe(3+)-MGD was used. Strong NO&z.rad; signals were measured from purified nNOS using the NO&z.rad; selective Fe(2+)-MGD and this was not affected by SOD. Thus, spin trapping with Fe-MGD can distinguish NO&z.rad; and NO(-) and this depends on the redox status of the iron. The detection of NO&z.rad; from purified NOS by Fe(2+)-MGD unambiguously reconfirms our previous report that NOS directly synthesizes NO&z.rad; but not NO(-).     

Radiophosphate (32-PO4) incorporation into phosphatidic acid of lead-poisoned and normalred cells.

Radiophosphate incorporation into phosphatidic acid is decreased in the red cells of lead-poisoned humans and rabbits. The decrease in activity is not related to the length of red cell survival nor to the differences in osmotic fragility observed in the lead-poisoned cells. Incorporation of radiophosphate into phosphatidic acid of normal red cells is sulfhydryl dependent and occurs at the cell surface. Lipid composition of the red cell membrane of the lead-poisoned humans and rabbits is normal.     

Enhanced ozone exposure of European beech (Fagus sylvatica) stimulates nitrogen mobilization from leaf litter and nitrogen accumulation in the soil

Abstract

In a lysimeter study with young beech trees, the effects of elevated ozone concentration on the decomposition and fate of nitrogen in ¹⁵N‐labeled leaf litter were analyzed after one growing season. Nitrogen in the litter was dominated by a relatively inert, residual fraction, but easily decomposable nitrogen was present in substantial amounts. Nitrogen loss was significantly higher at twice‐ambient ozone which was largely attributed to an enhanced mobilization of residual nitrogen. Enhanced mobilization of nitrogen from litter at twice‐ambient ozone exposure resulted in additional ¹⁵N incorporation into the soil down to 30 cm depth. Only 0.41–0.62% of the nitrogen in the litter was incorporated into plant material at both ozone concentrations. Twice‐ambient ozone exposure changed the distribution of the nitrogen taken up from litter inside the beech trees in favor of the shoot, where it may have been used in biosynthetic processes required for defense reactions.     

Identification and characterization of the iron regulatory element in the ferritin gene of a plant (soybean)

Iron increases ferritin synthesis, targeting plant DNA and animal mRNA. The ferritin promoter in plants has not been identified, in contrast to the ferritin promoter and mRNA iron-responsive element (IRE) in animals. The soybean leaf, a natural tissue for ferritin expression, and DNA, with promoter deletions and luciferase or glucuronidase reporters, delivered with particle bombardment, were used to show that an 86-base pair fragment (iron regulatory element (FRE)) controlled iron-mediated derepression of the ferritin gene. Mutagenesis with linkers of random sequence detected two subdomains separated by 21 base pairs. FRE has no detectable homology to the animal IRE or to known promoters in DNA and bound a trans-acting factor in leaf cell extracts. FRE/factor binding was abrogated by increased tissue iron, in analogy to mRNA (IRE)/iron regulatory protein in animals. Maximum ferritin derepression was obtained with 50 microm iron citrate (1:10) or 500 microm iron citrate (1:1) but Fe-EDTA was ineffective, although the leaf iron concentration was increased; manganese, zinc, and copper had no effect. The basis for different responses in ferritin expression to different iron complexes, as well as the significance of using DNA but not mRNA as an iron regulatory target in plants, remain unknown.     

A low-spin iron complex in human melanoma and rat hepatoma cells and a high-spin iron(II) complex in rat hepatoma cells.

Human melanoma and rat hepatoma cells cultured in the presence of low concentrations (2.5 microM) of low-molecular-weight iron (Fe) chelates and Fe-transferrin complexes have been studied with 57Fe M?ssbauer spectroscopy. The spectra show that holoferritin is only a minor fraction of the total iron present in the cells. The major form of Fe was in a low-spin state unlike the high-spin Fe(III) found in ferritin. Only about 10% of the Fe could be attributed to ferritin. In addition, the hepatoma cells had a high-spin Fe(II) spectral component which made up about 20% of the Fe present.     

Erythropoietin suppresses epithelial to mesenchymal transition and intercepts Smad signal transduction through a MEK-dependent mechanism in pig kidney (LLC-PK1) cell lines

Purpose

Tumor growth factor-β1 (TGF-β1) plays a pivotal role in processes like kidney epithelial-mesenchymal transition (EMT) and interstitial fibrosis, which correlate well with progression of renal disease. Little is known about underlying mechanisms that regulate EMT. Based on the anatomical relationship between erythropoietin (EPO)-producing interstitial fibroblasts and adjacent tubular cells, we investigated the role of EPO in TGF-β1-mediated EMT and fibrosis in kidney injury.

Methods

We examined apoptosis and EMT in TGF-β1-treated LLC-PK1 cells in the presence or absence of EPO. We examined the effect of EPO on TGF-β1-mediated Smad signaling. Apoptosis and cell proliferation were assessed with flow cytometry and hemocytometry. We used Western blotting and indirect immunofluorescence to evaluate expression levels of TGF-β1 signal pathway proteins and EMT markers.

Results

We demonstrated that ZVAD-FMK (a caspase inhibitor) inhibited TGF-β1-induced apoptosis but did not inhibit EMT. In contrast, EPO reversed TGF-β1-mediated apoptosis and also partially inhibited TGF-β1-mediated EMT. We showed that EPO treatment suppressed TGF-β1-mediated signaling by inhibiting the phosphorylation and nuclear translocation of Smad 3. Inhibition of mitogen-activated protein kinase kinase 1 (MEK 1) either directly with PD98059 or with MEK 1 siRNA resulted in inhibition of EPO-mediated suppression of EMT and Smad signal transduction in TGF-β1-treated cells.

Conclusions

EPO inhibited apoptosis and EMT in TGF-β1-treated LLC-PK1 cells. This effect of EPO was partially mediated by a mitogen-activated protein kinase-dependent inhibition of Smad signal transduction.     

The structure of d(CGCGAAT[]TCGCG) . d(CGCGAATTCGCG); the incorporation of a thymine photodimer into a B-DNA helix

In the light of the biological significance of thymine photodimers , studies of the energetics of the dodecanucleotide fragment d( CGCGAATTCGCG )2 have been carried out using the methods of molecular mechanics, with and without incorporation of a thymine dimer in the cis-syn configuration. The results of the calculations suggest that the thymine dimerized structures show no gross distortion in the double helix with the conformational changes relative to the normal B-DNA double helix restricted largely to the dimer region. The energetics of dTp[]dT reveal a number of conformers which are energetically almost equally favorable and are, as a group, qualitatively consistent with NMR studies on this molecule. The biological implications of the results of the conformational studies, reported here, have been examined vis-a-vis the currently available models for the recognition of DNA "damage" by repair enzymes.     

The role of iron nutrition in photosynthesis and nitrogen assimilation in SCENEDESMUS QUADRICAUDA (Chlorophyceae) 1

The rate of photosynthesis and nitrate uptake are related to the iron concentration in the medium for the green alga Scenedesmus quadricauda (Turp.) Breb. Increased iron leads to changes in chlorophyll a concentration, carbon fixation rate per chlorophyll a and in vivo fluorescence characteristics. These parameters all indicate that the efficiency of photosynthesis is related to iron nutrition. Nitrate uptake rate is also a function of both Fe and light Iron-limited cultures had decreased nitrate uptake at low light whereas ammonium uptake was relatively constant. Iron-limited cultures fixed about twice as much carbon into protein relative to the total carbon fixed. Iron plays a crucial role in the bioenergetics of carbon and nitrogen metabolism and may be important in controlling patterns of productivity.