Minocycline increases the life span and motor activity and decreases lipid peroxidation in manganese treated Drosophila melanogaster.

The objective of this study was to investigate the effect of Minocycline in the life span, motor activity, and lipid peroxidation of Drosophila melanogaster treated with manganese. Two days after emerging from the pupa male wild-type D. melanogaster were fed for 13 days with corn media containing 15 mM manganese. Then, they were divided in six groups of 300 flies each: group (a) remained treated with manganese (Mn group); group (b) began treatment with Minocycline (0.05 mM) (Mn-Minocycline group); group (c) received no additional treatment (Mn-no treatment group); group (d) simultaneously fed with manganese and Minocycline (Mn+Minocycline group). Additionally, a control (group e) with no treatment and another group (f) fed only with Minocycline after emerging from the pupa were added. All the manganese treated flies (group a) were dead on the 25th day. The life span in group f (101.66±1.33 days, mean S.E.M.) and of group b (97.00±3.46 days) were similar, but in both cases it was significantly higher than in group e (68.33±1.76 days), group c (67.05±2.30 days) and in those of group d (37.33±0.88). Manganese (groups a and d) decreased motor activity in D. melanogaster. In the Minocycline fed flies (groups b and f) a higher motor activity was detected. In Mn-Minocycline and Mn+Minocycline treated flies a significant decrease of MDA levels was detected when compared to the Minocycline group indicating that Minocycline and Mn appear to have a synergistic effect. In conclusion, Minocycline increased the life span and motor activity and decreased MDA formation of manganese treated D. melanogaster, probably by an inhibition of the production of reactive oxygen species. Manganese also exerted an antioxidant effect as shown by the significant decrease of MDA levels when compared to control flies.

Cross-resistance of cadmium-resistant cells to manganese is associated with reduced accumulation of both cadmium and manganese.

The mechanism of cellular entry of cadmium remains unclear. We have previously established cadmium-resistant cells from mouse embryonic cells of metallothionein (MT)-null mice, and demonstrated that the down-regulation of a zinc transporter, Zrt/Irt-related protein (ZIP) 8, was responsible for the reduced cadmium incorporation into cells. In the present study, we developed cadmium-resistant cells (A+70 and B+70) from mouse embryonic cells of MT-expressing wild-type mice. The LC50 values of CdCl2 for A+70 and B+70 cells were about 200 µM while that of the parental cells was 30 µM. We found that the cadmium resistance of these cells was conferred not only by enhanced expression of MT, but also by a decrease in cadmium accumulation. Since the uptake rates of cadmium into A+70 and B+70 cells were lowered, we determined the expression levels of the metal transporters and channels potentially involved in the cellular uptake of cadmium. We found a down-regulation of multiple transport systems, including ZIP8, divalent metal transporter 1 (DMT1), and α1 subunits of L-type (Ca(V)1.2) and T-type (Ca(V)3.1) voltage-dependent calcium channels, in A+70 and B+70 cells. Furthermore, A+70 and B+70 cells exhibited cross-resistance to cytotoxicity of MnCl2, probably due to a marked decrease in manganese uptake in these cells. These results suggest that the suppressed expression of ZIP8 and DMT1, which are known to have affinities for both cadmium and manganese, may be responsible for the reduction in the uptake, and consequently the cytotoxicity, of cadmium and manganese in A+70 and B+70 cells.

Estrogen and tamoxifen reverse manganese-induced glutamate transporter impairment in astrocytes.

Chronic exposure to manganese (Mn) can cause manganism, a neurodegenerative disorder similar to Parkinson's disease. The toxicity of Mn includes impairment of astrocytic glutamate transporters. 17beta-Estradiol (E2) has been shown to be neuroprotective in various neurodegenerative diseases including Parkinson's disease and Alzheimer's disease, and some selective estrogen receptor modulators, including tamoxifen (TX), also possess neuroprotective properties. We have tested our hypothesis that E2 and TX reverse Mn-induced glutamate transporter impairment in astrocytes. The results established that E2 and TX increased glutamate transporter function and reversed Mn-induced glutamate uptake inhibition, primarily via the up-regulation of glutamate/aspartate transporter (GLAST). E2 and TX also increased astrocytic GLAST mRNA levels and attenuated the Mn-induced inhibition of GLAST mRNA expression. In addition, E2 and TX effectively increased the expression of transforming growth factor beta1, a potential modulator of the stimulatory effects of E2/TX on glutamate transporter function. This effect was mediated by the activation of MAPK/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. These novel findings suggest, for the first time, that E2 and TX enhance astrocytic glutamate transporter expression via increased transforming growth factor beta1 expression. Furthermore, the present study is the first to show that both E2 and TX effectively reverse Mn-induced glutamate transport inhibition by restoring its expression and activity, thus offering a potential therapeutic modality in neurodegenerative disorders characterized by altered glutamate homeostasis.

BNIP3 up-regulation and mitochondrial dysfunction in manganese-induced neurotoxicity.

The central nervous system (CNS) appears to be the critical target of manganese (Mn), and neurotoxicity has been the focus of most of the health effects of manganese. In brain, the mechanism underlying the Mn-induced cell death is not clear. We have previously demonstrated that NFkappabeta induction and the activation of nitric oxide synthase through reactive oxygen species (ROS) represent a proximate mechanism for Mn-induced neurotoxicity. In this study, an immortalized dopaminergic cells were used to characterize the cell death signaling cascade activated by manganese. Exposure to Mn resulted in a time and concentration-related loss of cell viability as observed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and live/dead cell assay. Mn increased BNIP3 expression within 3h and continued to increase up to 24h exposure followed by a concentration-related apoptotic death as determined by TUNEL. Further, Mn treatment resulted in accumulation of reactive oxygen species and mitochondrial dysfunction with loss of mitochondrial membrane potential and release of cytochrome c. Antioxidants significantly reduced Mn-induced BNIP3 expression and attenuated cell death, demonstrating the role of oxidative stress in BNIP3 induction. Blocking BNIP3 up-regulation with a transcription or a translational inhibitor reduced the response to manganese. Cell death by manganese was reduced in the presence of CsA (PT pore inhibitor). In addition, knockdown of BNIP3 by small interfering RNA (siRNA) improved mitochondrial recovery and reduced neuronal cell loss suggesting that constitutive expression of BNIP3 plays a role in Mn-induced neurotoxicity by regulating mitochondrial functions. These findings indicate a potential detrimental role of BNIP3 in manganese-induced neuronal cell death.

Estrogen and tamoxifen protect against Mn-induced toxicity in rat cortical primary cultures of neurons and astrocytes.

Chronic exposure to manganese (Mn) leads to a neurological disorder, manganism, which shares multiple common features with idiopathic Parkinson disease (IPD). 17beta-Estradiol (E2) and some selective estrogen receptor modulators, including tamoxifen (TX), afford neuroprotection in various experimental models of neurodegeneration. However, the neuroprotective effects and mechanisms of E2/TX in Mn-induced toxicity have yet to be documented. Herein, we studied the ability of E2/TX to protect rat cortical primary neuronal and astroglial cultures from Mn-induced toxicity. Cell viability, Western blot, and reactive oxygen species (ROS) generation were assessed. Results established that both E2 (10nM) and TX (1 microM) attenuated Mn-induced toxicity. The protective effects of E2/TX were more pronounced in astrocytes versus neurons. The E2-mediated attenuation of Mn-induced ROS generation in astrocytes at 6-h treatment (where no cell death was detected) was mediated by a classical estrogen receptor (ER) pathway and the TX-mediated effect on Mn-induced ROS generation was not mediated via classical ER-dependent mechanisms and likely by its antioxidant properties. The phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway was involved in both E2- and TX-induced attenuation of Mn-induced ROS formation (6 h) in astrocytes. Treatments with Mn for a longer duration (24 h) led to significant cell death, and the protective effects of E2 and TX were (1) not mediated by a classical ER pathway and (2) associated with activation of both mitogen-activated protein kinase/extracellular signal-regulated kinase and PI3K/Akt signaling pathways. Taken together, the results suggest that both E2 and TX offer effective therapeutic means for neuroprotection against Mn-induced toxicity.

The effect of silicon on the symptoms of manganese toxicity in maize plants.

The effect of exogenously applied silicon (Si) on plant growth, lipid peroxidation, total phenolic compounds and non-protein thiols was studied in two maize varieties (Zea mays L. vars. Kneja 605, 434) differing in sensitivity to excess manganese (Mn). Based on the density of brown spots per leaf area and relative shoot weight (RSW) used to define Mn tolerance var. Kneja 434 was found to be more Mn-tolerant than Kneja 605. The lipid peroxidation level and total phenolic compounds were enhanced with increasing Mn concentration in the nutrient solution. In addition, the Mn-sensitive var. Kneja 605 with markedly expressed first visible Mn toxicity symptoms had higher levels of total phenolic acids than var. Kneja 434 thus supporting the hypothesis that a stimulating effect of Mn on phenol content reflected rather a stress response to Mn excess than a tolerance mechanism. In contrast, non-protein SH content increased to a higher extent in the Mn-tolerant var. Kneja 434. The increased amount of non-protein SH compounds was accompanied by a much stronger oxidative stress in the Mn-sensitive plants when compared with the Mn-tolerant variety, thus suggesting that non-protein SH compounds may play a role in Mn tolerance in maize. The addition of silicon (Si) reduced the density of brown spots per leaf area as well as lipid peroxidation level and improved plant growth in Mn-treated plants.

Synthesis of neuroprotective cyclopentenone prostaglandin analogs: suppression of manganese-induced apoptosis of PC12 cells.

The synthesis and evaluation for anti- and proapoptotic properties of cyclopentenone prostaglandin analogs are described. Novel J-type analogs of NEPP11 with a cross-conjugated cyclopentadienone moiety and a lipophilic omega-side chain suppressed manganese ion-induced apoptosis of PC12 cells at comparable levels to NEPP11, while monoenone derivatives were inactive. The proapoptotic activities of J-type analogs were much lower than that of NEPP11. Natural 15-deoxy-Delta(12,14)-PGJ(2) and Delta(7)-PGA(1) methyl ester were highly toxic, inducing apoptosis at lower concentrations.

Differential effect of nerve growth factor on dopaminergic neurotoxin-induced apoptosis.

Both rotenone and manganese are possible neurotoxins for a wide variety of cell and neuronal types including dopaminergic neurons and induce apoptosis in various cells. Neurotrophic factors have the potential for therapeutic development when used to prevent Parkinson's disease. In this paper, we focused on the differences between rotenone and manganese as toxins, and characterized the influence of neurotrophic factors on toxin-induced apoptosis in PC12 cells. There were distinct differences in intracellular mechanisms between rotenone- and manganese-induced apoptosis such as the production of reactive oxygen species, the response to antioxidants, and the activation of the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Nerve growth factor (NGF) almost completely prevented rotenone-induced but not manganese-induced caspase activation and DNA fragmentation. The differential effect of NGF was found to be mainly due to the down-regulation of the Trk tyrosine kinase receptor by manganese but not by rotenone. Prevention of rotenone-induced apoptosis by NGF was attenuated by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, but not MAPK kinase (MEK) inhibitors, PD98059 or U0126. These results demonstrate that the potential neurotoxins for dopaminergic cells exert their toxic effect by activation of different signaling pathways of apoptosis and that NGF prevents rotenone-induced apoptosis through the activation of the PI 3-kinase pathway not MAPK pathway.

Iron-blocking the high-affinity Mn-binding site in photosystem II facilitates identification of the type of hydrogen bond participating in proton-coupled electron transport via YZ.

Incubation of Mn-depleted PSII membranes [PSII(-Mn)] with Fe(II) is accompanied by the blocking of Y(Z)(*) at the high-affinity Mn-binding site to exogenous electron donors [Semin et al. (2002) Biochemistry 41, 5854-5864] and a shift of the pK(app) of the hydrogen bond partner for Y(Z) (base B) from 7.1 to 6.1 [Semin, B. K., and Seibert, M. (2004) Biochemistry 43, 6772-6782]. Here we calculate activation energies (E(a)) for Y(Z)(*) reduction in PSII(-Mn) and Fe-blocked PSII(-Mn) samples [PSII(-Mn, +Fe)] from temperature dependencies of the rate constants of the fast and slow components of the flash-probe fluorescence decay kinetics. At pH < pK(app) (e.g., 5.5), the decays are fit with one (fast) component in both types of samples, and E(a) is equal to 42.2 +/- 2.9 kJ/mol in PSII(-Mn) and 46.4 +/- 3.3 kJ/mol in PSII(-Mn, +Fe) membranes. At pH > pK(app), the decay kinetics exhibit an additional slow component in PSII(-Mn, +Fe) membranes (E(a) = 36.1 +/- 7.5 kJ/mol), which is much lower than the E(a) of the corresponding component observed for Y(Z)(*) reduction in PSII(-Mn) samples (48.1 +/- 1.7 kJ/mol). We suggest that the above difference results from the formation of a strong low barrier hydrogen bond (LBHB) between Y(Z) and base B in PSII(-Mn, +Fe) samples. To confirm this, Fe-blocking was performed in D(2)O to insert D(+), which has an energetic barrier distinct from H(+), into the LBHB. Measurement of the pH effects on the rates of Y(Z)(*) reduction in PSII(-Mn, +Fe) samples blocked in D(2)O shows a shift of the pK(app) from 6.1 to 7.6, and an increase in the E(a) of the slow component. This approach was also used to measure the stability of the Y(Z)(*) EPR signal at various temperatures in both kinds of membranes. In PSII(-Mn) membranes, the freeze-trapped Y(Z)(*) radical is stable below 190 K, but half of the Y(Z)(*) EPR signal disappears after a 1-min incubation when the sample is warmed to 253 K. In PSII(-Mn, +Fe) samples, the trapped Y(Z)(*) radical is unstable at a much lower temperature (77 K). However, the insertion of D(+) into the hydrogen bond between Y(Z) and base B during the blocking process increases the temperature stability of the Y(Z)(*) EPR signal at 77 K. Again, these results indicate that Fe-blocking involves Y(Z) in the formation of a LBHB, which in turn is consistent with the suggested existence of a LBHB between Y(Z) and base B in intact PSII membranes [Zhang, C., and Styring, S. (2003) Biochemistry 42, 8066-8076].

The effects of 17beta-estradiol and ethanol on zinc- or manganese-induced toxicity in SK-N-SH cells.

Serious neurodegenerative disorders are increasingly prevalent in our society and excessive oxidative stress may be a key mediator of neuronal cell death in many of these conditions. A variety of metals, such as manganese and zinc, are essential trace elements but can reach localized toxic concentrations through various disease processes or environmental exposures and have been implicated as having a role in neurodegeneration. Both manganese and zinc exist as bivalent cations and are essential cofactors/activators for numerous enzymes. Evidence suggests one action of these metals, when concentrated beyond physiological levels, may be to inhibit cellular energy production, ultimately leading to increased radical formation. Our studies were undertaken to directly investigate the toxic effects of manganese and zinc in an immortalized neuronal-like cell line (SK-N-SH) by testing interactions with the antioxidant, 17beta-estradiol, and the neurotoxin, ethanol. Employing undifferentiated SK-N-SH cells, we found that these metals caused biphasic effects, enhancing cell proliferation at low doses and inducing cell death at higher doses. Zinc was both more efficacious and more potent than manganese in enhancing growth and in causing cell death. 17beta-Estradiol and ethanol enhanced the proliferative actions of zinc and manganese across a wide concentration range. Furthermore, co-treatment with either 17beta-estradiol or ethanol afforded protection against manganese-, but not zinc-induced toxicity. Finally, combined administration of 17beta-estradiol and ethanol to SK-N-SH cells resulted in both a loss of growth enhancement and protective properties that were observed when these substances were administered individually. We also noted that the toxic effects occurred more rapidly from zinc than manganese exposure. Taken together, these data suggest that oxidative stress likely has a role in cell death resulting from toxic exposure to either zinc or manganese, but there is a difference in the precise mechanism of their effects.

Reduction-induced inhibition and Mn(II) release from the photosystem II oxygen-evolving complex by hydroquinone or NH2OH are consistent with a Mn(III)/Mn(III)/Mn(IV)/Mn(IV) oxidation state for the dark-adapted enzyme.

Hydroxylamine and hydroquinone were used to probe the oxidation states of Mn in the oxygen-evolving complex of dark-adapted intact (hydroxylamine) and salt-washed (hydroquinone) photosystem II. These preparations were incubated in the dark for 24 h in the presence of increasing reductant/photosystem II ratios, and the loss of oxygen evolution activity and of Mn(II) was determined for each incubation mixture. Monte Carlo simulations of these data yielded models that provide insight into the structure, reactivity, and oxidation states of the manganese in the oxygen-evolving complex. Specifically, the data support oxidation states of Mn(III)(2)/Mn(IV)(2) for the dark stable S(1) state of the O(2)-evolving complex. Activity and Mn(II) loss data were best modeled by assuming an S(1) --> S(-)(1) conversion of intermediate probability, a S(-)(1) --> S(-)(3) reaction of high probability, and subsequent step(s) of low probability. This model predicts that photosystem II Mn clusters that have undergone an initial reduction step become more reactive toward a second reduction, followed by a slower third reduction step. Analysis of the Mn(II) release parameters used to model the data suggests that the photosystem II manganese cluster consists of three Mn atoms that exhibit a facile reactivity with both reductants, and a single Mn that is reducible but sterically trapped at or near its binding site. Activity assays indicate that intact photosystem II centers reduced to S(-)(1) can evolve oxygen upon illumination, but that these centers are inactive in preparations depleted of the extrinsic 23 and 17 kDa polypeptides. Finally, it was found that a substantial population of the tyrosine D radical is reduced by hydroxylamine, but a smaller population reacts with hydroquinone over the course of a 24 h exposure to the reductant.

Direct oxidation of polymeric substrates by multifunctional manganese peroxidase isoenzyme from Pleurotus ostreatus without redox mediators.

VPs (versatile peroxidases) sharing the functions of LiP (lignin peroxidase) and MnP (manganese peroxidase) have been described in basidiomycetous fungi Pleurotus and Bjerkandera. Despite the importance of this enzyme in polymer degradation, its reactivity with polymeric substrates remains poorly understood. In the present study, we first report that, unlike LiP, VP from Pleurotus ostreatus directly oxidized two polymeric substrates, bovine pancreatic RNase and Poly R-478, through a long-range electron pathway without redox mediators. P. ostreatus produces several MnP isoenzymes, including the multifunctional enzyme MnP2 (VP) and a typical MnP isoenzyme MnP3. MnP2 (VP) depolymerized a polymeric azo dye, Poly R-478, to complete its catalytic cycle. Reduction of the oxidized intermediates of MnP2 (VP) to its resting state was also observed for RNase. RNase inhibited the oxidation of VA (veratryl alcohol) in a competitive manner. Blocking of the exposed tryptophan by N-bromosuccinimide inhibited the oxidation of RNase and VA by MnP2 (VP), but its Mn2+-oxidizing activity was retained, suggesting that Trp-170 exposed on an enzyme surface is a substrate-binding site both for VA and the polymeric substrates. The direct oxidation of RNase and Poly R by MnP2 (VP) is in sharp contrast with redox mediator-dependent oxidation of these polymers by LiP from Phanerochaete chrysosporium. Molecular modelling of MnP2 (VP) revealed that the differences in the dependence on redox mediators in polymer oxidation by MnP2 (VP) and LiP were explained by the anionic microenvironment surrounding the exposed tryptophan.

Magnesium ions alleviate the negative effect of manganese on Glomus claroideum BEG23.

The phytotoxicity of excessive levels of manganese (Mn), an essential micronutrient, can be alleviated significantly by a high supply of magnesium (Mg) ions to plants. A similar interaction of these two elements in the development of arbuscular mycorrhizal (AM) fungi has been verified in two experimental systems. In in vitro experiments, an outgrowth of hyphae from excised, surface-disinfected root segments colonised with Glomus claroideum BEG23 was measured after 5 days incubation in liquid media. When only Mn ions were present in the media at higher concentrations (>0.05 mM), the growth of hyphae from root segments was reduced significantly. Addition of magnesium sulphate to the incubation solution reduced the inhibitory effects of Mn on hyphal growth. Alleviation of Mn toxicity by Mg ions observed in in vitro experiments was verified also for the symbiotic association between G. claroideum and maize as a host plant in a hydroponics sand culture experiment.

Oxidative stress involves in astrocytic alterations induced by manganese.

It is hypothesized that manganese neurotoxicity could be secondary to a diminution of cellular protective and scavenger mechanisms. Since manganese is known to be sequestered in glial cells, we investigated possible neurotoxic mechanisms involving astrocytes in vitro. Astrocytes differentiated into process-bearing stellate cells in response to manganese treatment. Manganese concentration dependently decreased cellular DNA synthesis, glial fibrillary acidic protein expression, energy production, antioxidant capacity, and glutamate transporter activity. In contrast, manganese increased glutamine synthetase protein expression and cytokine-stimulated interleukin 6 mRNA expression. Under the concentration of 0.1 mM, manganese chloride caused no significant astrocyte death even up to 48 h after treatment. That is, these astrocytic alterations proceeded before the onset of cell demise. As a possible mediator of manganese-derived alterations, we determined intracellular redox state in astrocytes. Manganese time-dependently changed intracellular redox potential into oxidized state. The influx of manganese and its resultant oxidative stress was essential to most of the alterations, except for the action on stellation. Astrocytes are central component of the brain's antioxidant defense. Therefore, the observations suggest that dysfunction of astrocytes possibly involved in neurotoxic action of manganese.

Effect of the iron chelator desferrioxamine on manganese-induced toxicity of rat pheochromocytoma (PC12) cells.

Alterations in iron levels are likely to influence the biological actions of Mn in PC12 cells, because both metals are transported via the divalent metal transporter 1 (DMT1; also Nramp2 or DCT1). Studies were performed to determine the effect of the iron chelator desferrioxamine (DfO) on Mn-induced PC12 cell death and neuronal differentiation. Cell death almost doubled when PC12 cells were exposed for 24 hr to both DfO (10 microM) and Mn (0.3 mM) as opposed to Mn alone. DfO also stimulated Mn-induced neuronal differentiation by enhancing the phosphorylation of both ERK1 and 2 and also attenuated the increase in caspase 3-like activity induced by 0.3 mM Mn by approximately 50%, indicating that caspase activation, as reported previously, does not contribute to Mn-induced PC12 cell death. DfO also affected Mn-induced suppression of mitochondrial function as indicated by an additional 16% loss of ATP formation in PC12 cells cotreated with 0.3 mM Mn. Because sequestration of iron by DfO would be expected to lead to increased transport of Mn, studies were performed to determine whether iron inhibited Mn transport in PC12 cells. Iron inhibited 54Mn transport with an IC50 of approximately 20 microM. In addition, coincubation of DfO with Mn in PC12 cells resulted in increased expression of both the iron response element-positive and the iron response element-negative forms of DMT1. Taken together, these results demonstrate that iron status is likely to have a direct effect on the uptake and biological actions of Mn and probably other divalent metals that are transported by DMT1.

The regulation of integrin function by Ca(2+).

Integrins are metalloproteins whose receptor function is dependent on the interplay between Mg(2+) and Ca(2+). Although the specificity of the putative divalent cation binding sites has been poorly understood, some issues are becoming clearer and this review will focus on the more recent information. The MIDAS motif is a unique Mg(2+)/Mn(2+) binding site located in the integrin alpha subunit I domain. Divalent cation bound at this site has a structural role in coordinating the binding of ligand to the I domain containing integrins. The I-like domain of the integrin beta subunit also has a MIDAS-like motif but much less is known about its cation binding preferences. The N-terminal region of the integrin alpha subunit has been modelled as a beta-propeller, containing three or four 'EF hand' type divalent cation binding motifs for which the function is ill defined. It seems certain that most integrins have a high affinity Ca(2+) site which is critical for alphabeta heterodimer formation, but the location of this site is unknown. Finally intracellular Ca(2+) fluxes activate the Ca(2+) requiring enzyme, calpain, which regulates cluster formation of leucocyte integrins.

Divalent cations stimulate preferential recognition of a viral DNA end by HIV-1 integrase.

In the presence of a divalent metal cofactor (Mg2+ or Mn2+), retroviral-encoded integrase (IN) catalyzes two distinct reactions: site-specific cleavage of two nucleotides from both 3' ends of viral DNA, and sequence-independent joining of the recessed viral ends to staggered phosphates in a target DNA. Here we investigate human immunodeficiency virus type 1 (HIV-1) IN-DNA interactions using surface plasmon resonance. The results show that IN forms tight complexes both with duplex oligonucleotides that represent the viral DNA ends and with duplex oligonucleotides with an unrelated sequence that represent a target DNA substrate. The IN-DNA complexes are stable in 4.0 M NaCl, or 50% (v/v) methanol, but they are not resistant to low concentrations of SDS, indicating that their stability is highly dependent on structural features of the protein. Divalent metal cofactors exert two distinct effects on the IN-DNA interaction. Mn2+ inhibits IN binding to a model target DNA with the apparent Kd increasing approximately 3-fold in the presence of this cation. On the other hand, Mn2+ (or Mg2+) stimulates the binding of IN to a model viral DNA end, decreasing the apparent Kd of this IN-viral DNA complex approximately 6-fold. Such metal-mediated stimulation of the binding of IN to the viral DNA is totally abolished by substitution of the subterminal conserved CA/GT bp with a GT/CA bp, and is greatly diminished when the viral DNA end is recessed or "pre-processed." IN binds to a viral duplex oligonucleotide whose end was extended with nonviral sequences with kinetics similar to the nonviral model target DNA. This suggests that IN can distinguish the integrated DNA product from the viral donor DNA in the presence of divalent metal ion. Thus, our results show that preferential recognition of viral DNA by HIV-1 IN is achieved only in the presence of metal cofactor, and requires a free, wild-type viral DNA end.

Remarkable affinity and selectivity for Cs+ and uranyl (UO22+) binding to the manganese site of the apo-water oxidation complex of photosystem II.

The size and charge density requirements for metal ion binding to the high-affinity Mn2+ site of the apo-water oxidizing complex (WOC) of spinach photosystem II (PSII) were studied by comparing the relative binding affinities of alkali metal cations, divalent metals (Mg2+, Ca2+, Mn2+, Sr2+), and the oxo-cation UO22+. Cation binding to the apo-WOC-PSII protein was measured by: (1) inhibition of the rate and yield of photoactivation, the light-induced recovery of O2 evolution by assembly of the functional Mn4Ca1Clx, core from its constituent inorganic cofactors (Mn2+, Ca2+, and Cl-); and by (2) inhibition of the PSII-mediated light-induced electron transfer from Mn2+ to an electron acceptor (DCIP). Together, these methods enable discrimination between inhibition at the high- and low-affinity Mn2+ sites and the Ca2+ site of the apo-WOC-PSII. Unexpectedly strong binding of large alkali cations (Cs+ >> Rb+ > K+ > Na+ > Li+) was found to smoothly correlate with decreasing cation charge density, exhibiting one of the largest Cs+/Li+ selectivities (>/=5000) for any known chelator. Both photoactivation and electron-transfer measurements at selected Mn2+ and Ca2+ concentrations reveal that Cs+ binds to the high-affinity Mn2+ site with a slightly greater affinity (2-3-fold at pH 6.0) than Mn2+, while binding about 10(4)-fold more weakly to the Ca2+-specific site required for reassembly of functional O2 evolving centers. In contrast to Cs+, divalent cations larger than Mn2+ bind considerably more weakly to the high-affinity Mn2+ site (Mn2+ >> Ca2+ > Sr2+). Their affinities correlate with the hydrolysis constant for formation of the metal hydroxide by hydrolysis of water: Me2+aq --> [MeOH]+aq + H+aq. Along with the strong stimulation of the rate of photoactivation by alkaline pH, these metal cation trends support the interpretation that [MnOH]+ is the active species that forms upon binding of Mn2+aq to apo-WOC. Further support for this interpretation is found by the unusually strong inhibition of Mn2+ photooxidation by the linear uranyl cation (UO22+). The intrinsic binding constant for [MnOH]+ to apo-WOC was determined using a thermodynamic cycle to be K = 4.0 x 10(15) M-1 (at pH 6.0), consistent with a high-affinity, preorganized, multidentate coordination site. We propose that the selectivity for binding [MnOH]+, a linear low charge-density monocation, vs symmetrical Me2+ dications is functionally important for assembly of the WOC by enabling: (1) discrimination against higher charge density alkaline earth cations (Mg2+ and Ca2+) and smaller alkali metal cations (Na+ and K+) that are present in considerably greater abundance in vivo, and thus would suppress photoactivation; and (2) higher affinity binding of the one Ca2+ ion or the remaining three Mn2+ ions via coordination to form mu-hydroxo-bridged intermediates, apo-WOC-[Mn(mu-OH)2Mn]3+ or apo-WOC-[Mn(mu-OH)Ca]3+, during subsequent assembly steps of the native Mn4Ca1Clx core. In contrast to more acidic Me2+ divalent ion inhibitors of the high-affinity Mn2+ site, like Ca2+ and Sr2+, Cs+ does not accelerate the decay of the first light-induced intermediate, IM1, formed during photoactivation (attributed to apo-WOC-[Mn(OH)2]+). The inability of Cs+ to promote decay of IM1, despite having comparable affinity as Mn2+, is consistent with its considerably weaker Lewis acidity, resulting in the reprotonation of IM1 by water becoming the rate-limiting step for decay prior to displacement of Mn2+. All four different lines of evidence provide a self-consistent picture indicating that the initial step in assembly of the WOC involves high-affinity binding of [MnOH]+.

Manganese inhibits mitochondrial aconitase: a mechanism of manganese neurotoxicity.

The symptoms of Mn-induced neurotoxicity resemble those of Parkinson's diseases. Since iron (Fe) appears to play a pivotal role in pathophysiology of Parkinson's disease, we set out to test the hypothesis that alterations in Fe-requiring enzymes such as aconitase contribute to Mn-induced neurotoxicity. Mitochondrial fractions prepared from rat brain were preincubated with MnCl2 in vitro, followed by the enzyme assay. Mn treatment significantly inhibited mitochondrial aconitase activity (24% inhibition at 625 microM to 81% at 2.5 mM, p<0.05). The inhibitory effect was reversible and Mn-concentration dependent, and was reversed by the addition of Fe (0.05-1 mM) to the reaction mixture. In an in vivo chronic Mn exposure model, rats received intraperitoneal injection of 6 mg/kg Mn as MnCl2 once daily for 30 consecutive days. Mn exposure led to a region-specific alteration in total aconitase (i.e. , mitochondrial+cytoplasmic): 48.5% reduction of the enzyme activity in frontal cortex (p<0.01), 33.7% in striatum (p<0.0963), and 20.6% in substantia nigra (p<0.139). Chronic Mn exposure increased Mn concentrations in serum, CSF, and brain tissues. The elevation of Mn in all selected brain regions (range between 3.1 and 3.9 fold) was similar in magnitude to that in CSF (3.1 fold) rather than serum (6. 1 fold). The present results suggest that Mn alters brain aconitase activity, which may lead to the disruption of mitochondrial energy production and cellular Fe metabolism in the brain.

Mn(2+)-induced transient contraction of the longitudinal muscle of guinea-pig stomach through prostaglandin synthesis.

1. A low concentration of Mn2+ (less than 0.3 mM) transiently enhanced a contractile force (Mn(2+)-induced TC) of the longitudinal muscles of the guinea-pig stomach. 2. The Mn(2+)-induced transient contraction (TC) was not blocked by TTX (10(-7) M) or atropine (10(-6) M), nor by nifedipine (10(-6) M) or D-600 (10(-6) M), but was blocked by Ca2+ removal from the Krebs solution. 3. A preapplication of indomethacin (10(-7) M) completely inhibited an induction of the Mn(2+)-induced TC, but exogenous PGE2 (10(-7) M) was able to induce Mn(2+)-induced TC even with the presence of indomethacin (10(-7) M) and Mn2+ (0.1 mM) in the Krebs solution. 4. Quinacrine (10(-5) M), a phospholipase A2 inhibitor, partially inhibited the Mn(2+)-induced TC. 5. These results suggest that Mn(2+)-induced TC is probably mediated through cyclooxygenase and the subsequent generation of prostaglandin leading to the contraction.