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Manganese (Mn) is a trace nutrient necessary for life but becomes neurotoxic at high concentrations in the brain. The brain is a "privileged" organ that is separated from systemic blood circulation mainly by two barriers. Endothelial cells within the brain form tight junctions and act as the blood-brain barrier (BBB), which physically separates circulating blood from the brain parenchyma. Between the blood and the cerebrospinal fluid (CSF) is the choroid plexus (CP), which is a tissue that acts as the blood-CSF barrier (BCB). Pharmaceuticals, proteins, and metals in the systemic circulation are unable to reach the brain and spinal cord unless transported through either of the two brain barriers. The BBB and the BCB consist of tightly connected cells that fulfill the critical role of neuroprotection and control the exchange of materials between the brain environment and blood circulation. Many recent publications provide insights into Mn transport in vivo or in cell models. In this review, we will focus on the current research regarding Mn metabolism in the brain and discuss the potential roles of the BBB and BCB in maintaining brain Mn homeostasis.

Common genetic variants interact with environmental factors to impact risk of heritable diseases. A notable example of this is a single-nucleotide variant in the Solute Carrier Family 39 Member 8 ( SLC39A8 ) gene encoding the missense variant A391T, which is associated with a variety of traits ranging from Parkinson's disease and neuropsychiatric disease to cardiovascular and metabolic diseases and Crohn's disease. The remarkable extent of pleiotropy exhibited by SLC39A8 A391T raises key questions regarding how a single coding variant can contribute to this diversity of clinical outcomes and what is the mechanistic basis for this pleiotropy. Here, we generate a murine model for the Slc39a8 A391T allele and demonstrate that these mice exhibit Mn deficiency in the colon associated with impaired intestinal barrier function and epithelial glycocalyx disruption. Consequently, Slc39a8 A391T mice exhibit increased sensitivity to epithelial injury and pathological inflammation in the colon. Taken together, our results link a genetic variant with a dietary trace element to shed light on a tissue-specific mechanism of disease risk based on impaired intestinal barrier integrity., Competing Interests: Competing interest statement: R.J.X. is cofounder of Celsius Therapeutics and Jnana Therapeutics. These companies did not provide support for this work., (Copyright © 2020 the Author(s). Published by PNAS.)

Metals are essential components in all forms of life required for the function of nearly half of all enzymes and are critically involved in virtually all fundamental biological processes. Especially, the transition metals iron (Fe), zinc (Zn), manganese (Mn), nickel (Ni), copper (Cu) and cobalt (Co) are crucial micronutrients known to play vital roles in metabolism as well due to their unique redox properties. Metals carry out three major functions within metalloproteins: to provide structural support, to serve as enzymatic cofactors, and to mediate electron transportation. Metal ions are also involved in the immune system from metal allergies to nutritional immunity. Within the past decade, much attention has been drawn to the roles of metal ions in the immune system, since increasing evidence has mounted to suggest that metals are critically implicated in regulating both the innate immune sensing of and the host defense against invading pathogens. The importance of ions in immunity is also evidenced by the identification of various immunodeficiencies in patients with mutations in ion channels and transporters. In addition, cancer immunotherapy has recently been conclusively demonstrated to be effective and important for future tumor treatment, although only a small percentage of cancer patients respond to immunotherapy because of inadequate immune activation. Importantly, metal ion-activated immunotherapy is becoming an effective and potential way in tumor therapy for better clinical application. Nevertheless, we are still in a primary stage of discovering the diverse immunological functions of ions and mechanistically understanding the roles of these ions in immune regulation. This review summarizes recent advances in the understanding of metal-controlled immunity. Particular emphasis is put on the mechanisms of innate immune stimulation and T cell activation by the essential metal ions like calcium (Ca 2+ ), zinc (Zn 2+ ), manganese (Mn 2+ ), iron (Fe 2+ /Fe 3+ ), and potassium (K + ), followed by a few unessential metals, in order to draw a general diagram of metalloimmunology., (© 2020 Elsevier Inc. All rights reserved.)

Divalent metals such as iron and manganese play an important role in the cellular response to oxidative challenges and are required as cofactors by many enzymes. However, how these metals affect replication after oxidative challenge is not known. Here, we show that replication in Escherichia coli is inhibited following a challenge with hydrogen peroxide and requires manganese for the rapid recovery of DNA synthesis. We show that the manganese-dependent recovery of DNA synthesis occurs independent of lesion repair, modestly improves cell survival, and is associated with elevated rates of mutagenesis. The Mn-dependent mutagenesis involves both replicative and translesion polymerases and requires prior disruption by H 2 O 2 to occur. Taking these findings together, we propose that replication in E. coli is likely to utilize an iron-dependent enzyme(s) that becomes oxidized and inactivated during oxidative challenges. The data suggest that manganese remetallates these or alternative enzymes to allow genomic DNA replication to resume, although with reduced fidelity. IMPORTANCE Iron and manganese play important roles in how cell's cope with oxygen stress. However, how these metals affect the ability of cells to replicate after oxidative challenges is not known. Here, we show that replication in Escherichia coli is inhibited following a challenge with hydrogen peroxide and requires manganese for the rapid recovery of DNA synthesis. The manganese-dependent recovery of DNA synthesis occurs independently of lesion repair and modestly improves survival, but it also increases the mutation rate in cells. The results imply that replication in E. coli is likely to utilize an iron-dependent enzyme(s) that becomes oxidized and inactivated during oxidative challenges. We propose that manganese remetallates these or alternative enzymes to allow genomic DNA replication to resume, although with reduced fidelity., (Copyright © 2019 American Society for Microbiology.)

Purpose of Review: This article provides an overview of the pathogenesis, clinical presentation and treatment of inherited manganese transporter defects., Recent Findings: Identification of a new group of manganese transportopathies has greatly advanced our understanding of how manganese homeostasis is regulated in vivo. While the manganese efflux transporter SLC30A10 and the uptake transporter SLC39A14 work synergistically to reduce the manganese load, SLC39A8 has an opposing function facilitating manganese uptake into the organism. Bi-allelic mutations in any of these transporter proteins disrupt the manganese equilibrium and lead to neurological disease: Hypermanganesaemia with dystonia 1 (SLC30A10 deficiency) and hypermanganesaemia with dystonia 2 (SLC39A14 deficiency) are characterised by manganese neurotoxicity while SLC39A8 mutations cause a congenital disorder of glycosylation type IIn due to Mn deficiency. Inherited manganese transporter defects are an important differential diagnosis of paediatric movement disorders. Manganese blood levels and MRI brain are diagnostic and allow early diagnosis to avoid treatment delay.

Manganese is an essential dietary element that functions primarily as a coenzyme in several biological processes. These processes include, but are not limited to, macronutrient metabolism, bone formation, free radical defense systems, and in the brain, ammonia clearance and neurotransmitter synthesis. It is a critical component in dozens of proteins and enzymes, and is found in all tissues. Concentrated levels of Mn are found in tissues rich in mitochondria and melanin, with both, liver, and pancreas having the highest concentrations under normal conditions. However, overexposure to environmental Mn via industrial occupation or contaminated drinking water can lead to toxic brain Mn accumulation that has been associated with neurological impairment. The objective of this chapter is to address the biological importance of Mn (essentiality), routes of exposure, factors dictating Mn status, a brief discussion of Mn neurotoxicity, and proposed methods for neurotoxicity remediation.

Members of the cation diffusion facilitator (CDF) family have been identified in all kingdoms of life. They have been divided into three subgroups, namely Zn-CDF, Fe/Zn-CDF, and Mn-CDF, based on their putative specificity to transported metal ions. The plant metal tolerance protein 6 (MTP6) proteins fall into the Fe/Zn-CDF subgroup; however, their function in iron/zinc transport has not yet been confirmed. Here, we characterized the MTP6 protein from cucumber, Cucumis sativus. When expressed in yeast and in protoplasts isolated from Arabidopsis cells, CsMTP6 localized in mitochondria and contributed to the efflux of Fe and Mn from these organelles. Immunolocalization of CsMTP6 in cucumber membranes confirmed this association with mitochondria. Root expression and protein levels of CsMTP6 were significantly up-regulated in conditions of Fe deficiency and excess, but were not affected by Mn availability. These results indicate that MTP6 proteins contribute to the distribution of Fe and Mn between the cytosol and mitochondria of plant cells, and are regulated by Fe to maintain mitochondrial and cytosolic iron homeostasis under varying conditions of Fe availability.

To obtain near-IR absorbing biomaterials as fluorescence cellular imaging and anticancer agents for hypoxic cancer cell, a nano NIR fluorescence Mn(III/IV) polymer (PMnD) was spectroscopically characterized. The PMnD shows strong emission at 661nm when excited with 643nm. Furthermore, PMnD can catalyze water oxidation to generate dioxygen when irradiated by red LED light (10W). In particular, the PMnD can enter into HepG-2 cells and mitochondria. Both anticancer activity and the inhibition of the expression of HIF-1α for PMnD were concentration dependent. Our results demonstrate that PMnD can be developed as mitochondria targeted imaging agents and new inhibitors for HIF-1 in hypoxic cancer cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Manganese (Mn) is an essential micronutrient and required cofactor in bacteria. Despite its importance, excess Mn can impair bacterial growth, the mechanism of which remains largely unexplored. Here, we show that proper Mn homeostasis is critical for cellular growth of the major human respiratory pathogen Streptococcus pneumoniae. Perturbations in Mn homeostasis genes, psaBCA, encoding the Mn importer, and mntE, encoding the Mn exporter, lead to Mn sensitivity during aerobiosis. Mn-stressed cells accumulate iron and copper, in addition to Mn. Impaired growth is a direct result of Mn toxicity and does not result from iron-mediated Fenton chemistry, since cells remain sensitive to Mn during anaerobiosis or when hydrogen peroxide biogenesis is significantly reduced. Mn-stressed cells are significantly elongated, whereas Mn-limitation imposed by zinc addition leads to cell shortening. We show that Mn accumulation promotes aberrant dephosphorylation of cell division proteins via hyperactivation of the Mn-dependent protein phosphatase PhpP, a key enzyme involved in the regulation of cell division. We discuss a mechanism by which cellular Mn:Zn ratios dictate PhpP specific activity thereby regulating pneumococcal cell division. We propose that Mn-metalloenzymes are particularly susceptible to hyperactivation or mismetallation, suggesting the need for exquisite cellular control of Mn-dependent metabolic processes., (© 2017 John Wiley & Sons Ltd.)

Toxin-antitoxin (TA) systems play important roles in bacterial physiology, such as multidrug tolerance, biofilm formation, and arrest of cellular growth under stress conditions. To develop novel antimicrobial agents against tuberculosis, we focused on VapBC systems, which encompass more than half of TA systems in Mycobacterium tuberculosis. Here, we report that theMycobacterium tuberculosis VapC30 toxin regulates cellular growth through both magnesium and manganese ion-dependent ribonuclease activity and is inhibited by the cognate VapB30 antitoxin. We also determined the 2.7-Å resolution crystal structure of the M. tuberculosis VapBC30 complex, which revealed a novel process of inactivation of the VapC30 toxin via swapped blocking by the VapB30 antitoxin. Our study on M. tuberculosis VapBC30 leads us to design two kinds of VapB30 and VapC30-based novel peptides which successfully disrupt the toxin-antitoxin complex and thus activate the ribonuclease activity of the VapC30 toxin. Our discovery herein possibly paves the way to treat tuberculosis for next generation., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The cyanobacterium Anabaena sp. PCC 7120 was grown in presence and absence of iron to decipher the role of manganese in protection against the oxidative stress under iron starvation and growth, manganese uptake kinetics, antioxidative enzymes, lipid peroxidation, electrolyte leakage, thiol content, total peroxide, proline and NADH content was investigated. Manganese supported the growth of cyanobacterium Anabaena 7120 under iron deprived conditions where maximum uptake rate of manganese was observed with lower K(m) and higher V(max) values. Antioxidative enzymes were also found to be elevated in iron-starved conditions. Estimation of lipid peroxidation and electrolyte leakage depicted the role of manganese in stabilizing the integrity of the membrane which was considered as the prime target of oxygen free radicals in oxidative stress. The levels of total peroxide, thiol, proline and NADH content, which are the representative of oxidative stress response in Anabaena 7120, were also showed increasing trends in iron starvation. Hence, the results discerned, clearly suggested the role of manganese in protection against the oxidative stress in cyanobacterium Anabaena 7120 under iron starvation either due to its antioxidative properties or involvement as cofactor in a number of antioxidative enzymes., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The understanding of manganese (Mn) biology, in particular its cellular regulation and role in neurological disease, is an area of expanding interest. Mn is an essential micronutrient that is required for the activity of a diverse set of enzymatic proteins (e.g., arginase and glutamine synthase). Although necessary for life, Mn is toxic in excess. Thus, maintaining appropriate levels of intracellular Mn is critical. Unlike other essential metals, cell-level homeostatic mechanisms of Mn have not been identified. In this review, we discuss common forms of Mn exposure, absorption, and transport via regulated uptake/exchange at the gut and blood-brain barrier and via biliary excretion. We present the current understanding of cellular uptake and efflux as well as subcellular storage and transport of Mn. In addition, we highlight the Mn-dependent and Mn-responsive pathways implicated in the growing evidence of its role in Parkinson's disease and Huntington's disease. We conclude with suggestions for future focuses of Mn health-related research.

Research directed at anatomical, integrative and functional activities of the central nervous system (CNS) can be realized through bioimaging. A wealth of data now demonstrates the utility of magnetic resonance imaging (MRI) towards unraveling complex neural connectivity operative in health and disease. A means to improve MRI sensitivity is through contrast agents and notably manganese (Mn²⁺). The Mn²⁺ ions enter neurons through voltage-gated calcium channels and unlike other contrast agents such as gadolinium, iron oxide, iron platinum and imaging proteins, provide unique insights into brain physiology. Nonetheless, a critical question that remains is the brain target cells serving as sources for the signal of Mn²⁺ enhanced MRI (MEMRI). To this end, we investigated MEMRI's abilities to detect glial (astrocyte and microglia) and neuronal activation signals following treatment with known inflammatory inducing agents. The idea is to distinguish between gliosis (glial activation) and neuronal injury for the MEMRI signal and as such use the agent as a marker for neural activity in inflammatory and degenerative disease. We now demonstrate that glial inflammation facilitates Mn²⁺ neuronal ion uptake. Glial Mn²⁺ content was not linked to its activation. MEMRI performed on mice injected intracranially with lipopolysaccharide was associated with increased neuronal activity. These results support the notion that MEMRI reflects neuronal excitotoxicity and impairment that can occur through a range of insults including neuroinflammation. We conclude that the MEMRI signal enhancement is induced by inflammation stimulating neuronal Mn²⁺ uptake.

The Lyme disease pathogen Borrelia burgdorferi represents a novel organism in which to study metalloprotein biology in that this spirochete has uniquely evolved with no requirement for iron. Not only is iron low, but we show here that B. burgdorferi has the capacity to accumulate remarkably high levels of manganese. This high manganese is necessary to activate the SodA superoxide dismutase (SOD) essential for virulence. Using a metalloproteomic approach, we demonstrate that a bulk of B. burgdorferi SodA directly associates with manganese, and a smaller pool of inactive enzyme accumulates as apoprotein. Other metalloproteins may have similarly adapted to using manganese as co-factor, including the BB0366 aminopeptidase. Whereas B. burgdorferi SodA has evolved in a manganese-rich, iron-poor environment, the opposite is true for Mn-SODs of organisms such as Escherichia coli and bakers' yeast. These Mn-SODs still capture manganese in an iron-rich cell, and we tested whether the same is true for Borrelia SodA. When expressed in the iron-rich mitochondria of Saccharomyces cerevisiae, B. burgdorferi SodA was inactive. Activity was only possible when cells accumulated extremely high levels of manganese that exceeded cellular iron. Moreover, there was no evidence for iron inactivation of the SOD. B. burgdorferi SodA shows strong overall homology with other members of the Mn-SOD family, but computer-assisted modeling revealed some unusual features of the hydrogen bonding network near the enzyme's active site. The unique properties of B. burgdorferi SodA may represent adaptation to expression in the manganese-rich and iron-poor environment of the spirochete.

The role of transition metal ions in atherogenesis is controversial; they can participate in the hydroxyl radical generation and catalyze the reactive oxygen species neutralization reaction as cofactors of antioxidant enzymes. Using EPR spectroscopy, we revealed that 70% of the samples of aorta with atherosclerotic lesions possessed superoxide dismutase activity, 100% of the samples initiated Fenton reaction and demonstrated the presence of manganese paramagnetic centers. The sodA gene encoding manganese-dependent bacterial superoxide dismutase was not found in the samples of atherosclerotic plaques by PCR using degenerate primers. The data obtained indicates the perspectives of manganese analysis as a marker element in the express diagnostics of atherosclerosis.

Arsenate retention, arsenite sorption and oxidation on the surfaces of Fe-Mn binary oxides may play an important role in the mobilization and transformation of arsenic, due to the common occurrence of these oxides in the environment. However, no sufficient information on the sorption behaviors of arsenic on Fe-Mn binary oxides is available. This study investigated the influences of Mn/Fe molar ratio, solution pH, coexisting calcium ions, and humic acids have on arsenic sorption by Fe-Mn binary oxides. To create Fe-Mn binary oxides, simultaneous oxidation and co-precipitation methods were employed. The Fe-Mn binary oxides exhibited a porous crystalline structure similar to 2-line ferrihydrite at Mn/Fe ratios 1:3 and below, whereas exhibited similar structures to δ-MnO(2) at higher ratios. The As(V) sorption maximum was observed at a Mn/Fe ratio of 1:6, but As(III) uptake maximum was at Mn/Fe ratio 1:3. However, As(III) adsorption capacity was much higher than that of As(V) at each Mn/Fe ratio. As(V) sorption was found to decrease with increasing pH, while As(III) sorption edge was different, depending on the content of MnO(2) in the binary oxides. The presence of Ca(2+) enhanced the As(V) uptake under alkaline pH, but did not significantly influence the As(III) sorption by 1:9 Fe-Mn binary oxide; whereas the presence of humic acid slightly reduced both As(V) and As(III) uptake. These results indicate that As(III) is more easily immobilized than As(V) in the environment, where Fe-Mn binary oxides are available as sorbents and they represent attractive adsorbents for both As(V) and As(III) removal from water and groundwater., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Antlers represent an ideal experimental model for bone biology studies, because of their easy accessibility, and their rapid growth. Findings from our previous studies revealed that Mn plays an essential role in incorporating the circulating bone Ca to the growing antlers. Based on these findings, we hypothesize that Mn, an essential mineral for Ca fixation (or incorporation) into bones, might be released from bone, during its remodeling, to be available for prioritized function, most likely, brain function; Consequently, Ca incorporation will be dramatically affected, leading to osteoporosis, particularly in elderly people. Therefore, osteoporosis would precede brain malfunctioning diseases such as Alzheimer's or Parkinson's, and clinical data are available to support some of the predictions derived from this hypothesis.

This study further investigates the influence of temporarily disrupting the blood-brain barrier (BBB) on the level of manganese used in AIM fMRI other than the recognized function of allowing that substance to enter into the activated brain regions more effectively during the BBB opening. We injected manganese into Wistar rats through ICA following the disruption of BBB with mannitol in a functional MRI test of the visual cortex. Through comparing MRI signal intensity and manganese contents in the visual cortex of rats received visual stimuli of unequal degree after the restoration of BBB, we found that the signal in the visual cortex could be further enhanced on T1WI given visual stimulation after the restoration of BBB. Temporary BBB disruption has an additional advantage in allowing Mn(2+) to enter the CSF or brain for later transference to the activated brain area. So the dosage of manganese in AIM fMRI could be minimized by extending the stimulus.

Manganese is an important element essential for human functioning. Pathogenesis of manganese intoxication remains unclear. Specification of differential diagnostic criteria is required for diagnosis of occupational manganese intoxication and ruling out Parkinson disease and secondary parkinsonism in the patients.

Manganese is an essential trace element but its overexposure causes poisoning (called manganism) that shares several symptoms with Parkinson's disease, but with a mechanism that is still not well understood: in addition to involvement of the dopaminergic system, both serotonergic and peptiergic systems have been implicated. In the present report we have studied the influence of Mn(2+) on 5-HT(1A) receptor signaling complexes in rat brain and found that Mn(2+) in millimolar concentration caused an increase of high-affinity agonist binding to rat hippocampal membranes in comparison with experiments in the presence of Mg(2+), but not in rat cortical membranes and in Sf9 cell membranes expressing 5-HT(1A) receptors and G(i1) heterotrimers. Activation of G proteins with 30μM GTPγS turned all 5-HT(1A) receptors in these preparations into a low-affinity state for agonist binding in the presence of 1mM Mg(2+), but not in the presence of 1mM Mn(2+) in rat hippocampal membranes. However, if 1μM GTPγS was used for G protein activation, a substantial amount of high affinity agonist binding was detected in the presence of Mn(2+) also in cortical membranes and Sf9 cells, but not with Mg(2+) or EDTA. Comparison of the abilities of GDP and GTPγS to modulate high affinity agonist binding to 5-HT(1A) receptors indicated that both nucleotides were almost 10-fold less potent in the presence of MnCl(2) compared to MgCl(2). This means that by inhibiting guanosine nucleotide binding to G proteins in complex with 5-HT(1A) receptors, Mn(2+) acts as an enhancer for agonist binding and signal transduction. As the influence of Mn(2+) resembles the hypersensitivity of dopaminergic system in Parkinsonial models, it can be proposed that at least some symptoms of manganism are connected with a change of signal transduction complex caused by manganese-nucleotide complexes., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Bradyrhizobium japonicum Fur mediates manganese-responsive transcriptional control of the mntH gene independently of iron, but it also has been implicated in iron-dependent regulation of the irr gene. Thus, we sought to address the apparent discrepancy in Fur responsiveness to metals. Irr is a transcriptional regulator found in iron-limited cells. Here, we show that irr gene mRNA was regulated by both iron and manganese, and repression occurred only in the presence of both metals. Under these conditions, Fur occupied the irr promoter in vivo in the parent strain, and irr mRNA expression was derepressed in a fur mutant. Under low iron conditions, the irr promoter was occupied by Irr, but not by Fur, and control by manganese was lost. Fur occupancy of the irr promoter was dependent on manganese, but not iron, in an irr mutant, suggesting that Irr normally interferes with Fur binding. Correspondingly, regulation of irr mRNA was dependent only on manganese in the irr strain. The Irr binding site within the irr promoter partially overlaps the Fur binding site. DNase I footprinting analysis showed that Irr interfered with Fur binding in vitro. In addition, Fur repression of transcription from the irr promoter in vitro was relieved by Irr. We conclude that Fur mediates manganese-dependent repression of irr transcription and that Irr acts as an antirepressor under iron limitation by preventing Fur binding to the promoter.

Manganese (Mn) and iron (Fe) are transition metals that are crucial to the appropriate growth, development, function, and maintenance of biological organisms. Because of their chemical similarity, in organisms ranging from bacteria to mammals they share and compete for many protein transporters, such as the divalent metal transporter-1. As such, during conditions of low Fe, abnormal Mn accumulation occurs. Conversely, when Mn concentrations are altered, the homeostasis and deposition of Fe and other transition metals are disrupted. Our lab has undertaken a series of studies in rats involving pregnant dams, neo- and perinatal pups, and adult animals. Animals were exposed to various concentrations of dietary Fe and/or Mn, and protein transporter expression, blood Mn and Fe concentrations, brain transition metal concentrations, and temporal brain deposition patterns were examined. As a result, we have demonstrated the importance of the interdependence of the transport of Mn and Fe, and established brain metal concentrations in several longitudinal studies. The purpose of this review is to examine these studies in their entirety and highlight the importance of monitoring the deposition and accumulation of both Mn and Fe when designing future studies related to either dietary or environmental changes in transition metal levels. Finally, this review will provide information about various transport proteins currently under investigation in the research community related to Fe and Mn regulation and transport.

Perception and response to nutritional iron availability by bacteria are essential to control cellular iron homeostasis. The Irr protein from Bradyrhizobium japonicum senses iron through the status of heme biosynthesis to globally regulate iron-dependent gene expression. Heme binds directly to Irr to trigger its degradation. Here, we show that severe manganese limitation created by growth of a Mn(2+) transport mutant in manganese-limited media resulted in a cellular iron deficiency. In wild-type cells, Irr levels were attenuated under manganese limitation, resulting in reduced promoter occupancy of target genes and altered iron-dependent gene expression. Irr levels were high regardless of manganese availability in a heme-deficient mutant, indicating that manganese normally affects heme-dependent degradation of Irr. Manganese altered the secondary structure of Irr in vitro and inhibited binding of heme to the protein. We propose that manganese limitation destabilizes Irr under low-iron conditions by lowering the threshold of heme that can trigger Irr degradation. The findings implicate a mechanism for the control of iron homeostasis by manganese in a bacterium.

Mice with experimental neuropsychiatric lupus (NPSLE), induced by anti-ribosomal-P antibodies, developed depression-like behavior and a diminished sense of smell. Manganese-enhanced MRI (MEMRI) allows in vivo mapping of functional neuronal connections in the brain, including the olfactory tract. The aim of this study was to analyze and describe, via the MEMRI technique, the effect of the anti-ribosomal-P injection on the olfactory pathway. Twenty mice were intra-cerebra-ventricular injected to the right hemisphere: 10 with human anti-ribosomal-P antibodies and 10 with human IgG antibodies (control). Depression was addressed by forced swimming test and smell function was evaluated by smelling different concentrations of menthol. MEMRI was used to investigate the olfactory system in these mice. Passive transfer of anti-ribosomal-P to mice resulted in a depression-like behavior, accompanied with a significant deficit in olfactory function. MEMRI of these mice demonstrated significant reduction (P < 0.001) in normalized manganese enhancement ratios of olfactory structures, compared to control mice. We concluded that an impaired olfactory neuronal function in mice with experimental depression, mediated by passive transfer of human-anti-ribosomal-P, can be demonstrated by MEMRI.

In the manganese-depleted photosystem II (PSII[-Mn]) preparations, oxidation of exogenous electron donors is carried out through the high-affinity (HA) and the low-affinity (LA) sites. This paper investigates the LA oxidation site in the PSII(-Mn) preparations where the HA, Mn-binding site was blocked with ferric cations [[11] B.K. Semin, M.L. Ghirardi, M. Seibert, Blocking of electron donation by Mn(II) to Y(Z)(*) following incubation of Mn-depleted photosystem II membranes with Fe(II) in the light, Biochemistry 41 (2002) 5854-5864.]. In blocked (PSII[-Mn,+Fe]) preparations electron donation by Mn(II) cations to Y(Z)(*) was not detected at Mn(II) concentration 10 microM (corresponds to K(m) for Mn(II) oxidation at the HA site), but detected at Mn concentration 100 microM (corresponds to K(m) for the LA site) by fluorescence measurements. Comparison of pH-dependencies of electron donation by Mn(II) through the HA and the LA sites revealed the similar pK(a) equal to 6.8. Comparison of K(m) for diphenylcarbazide (DPC) oxidation at the LA site and K(d) for A(T) thermoluminescence band suppression by DPC in PSII(-Mn,+Fe) samples suggests that there is relationship between the LA site and A(T) band formation. The role of D1-His190 as an oxidant of exogenous electron donors at the LA site is discussed. In contrast to electrogenic electron transfer from Mn(II) at the HA site to Y(Z)(*), photovoltage due to Mn(II) oxidation in iron-blocked PSII(-Mn) core particles was not detected.

Chronic exposure to manganese causes parkinsonian symptoms and has been implicated as an environmental factor in the pathogenesis of Parkinson's disease (PD). Here we show that manganese inhibits the proliferation of PC12 cells and induces apoptosis through the formation of catechol isoquinolines. Manganese induces the production of 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol, Sal) and N-methyl-salsolinol (NMSal) in PC12 cells, and increases the levels of malondialdehyde (MDA) in a dose-dependent manner. The data indicates that the formation of catechol isoquinolines due to oxidative stress induced by MnCl(2) may be a mechanism by which manganese causes degeneration of dopaminergic neurons.

Manganese (Mn) is an essential element present in all living organisms and is naturally present in rocks, soil, water, and food. Exposure to high oral, parenteral, or ambient air concentrations of Mn can result in elevations in Mn tissue levels and neurological effects. However, current understanding of the impact of Mn exposure on the nervous system leads to the hypothesis that there should be no adverse effects at low exposures, because Mn is an essential element; therefore, there should be some threshold for exposure above which adverse effects may occur and adverse effects may increase in frequency with higher exposures beyond that threshold. Data gaps regarding Mn neurotoxicity include what the clinical significance is of the neurobehavioural, neuropsychological, or neurological endpoints measured in many of the occupational studies that have evaluated cohorts exposed to relatively low levels of Mn. Specific early biomarkers of effect, such as subclinical neurobehavioural or neurological changes or magnetic resonance imaging (MRI) changes have not been established or validated for Mn, although some studies have attempted to correlate biomarkers with neurological effects. Experimental studies with rodents and monkeys provide valuable information about the absorption, bioavailability, and tissue distribution of various Mn compounds with different solubilities and oxidation states in different age groups. Studies have shown that rodents and primates maintain stable tissue manganese levels as a result of homeostatic mechanisms that tightly regulate absorption and excretion. In addition, physiologically based pharmacokinetic (PBPK) models are being developed to provide for the ability to conduct route-to-route extrapolations, evaluate nasal uptake to the CNS, and evaluate lifestage differences in Mn pharmacokinetics. Such models will facilitate more rigorous quantitative analysis of the available pharmacokinetic data for Mn and will be used to identify situations that may lead to increased brain accumulation related to altered Mn metabolism in different human populations, and develop quantitatively accurate predictions of increased Mn levels that may serve as a basis of dosimetry-based risk assessments. Such assessments will permit for the development of more scientifically refined and robust recommendations, guidelines, and regulations for Mn levels in the ambient environment and occupational settings.

The apoplast is known to play a predominant role in the expression of manganese (Mn) toxicity in cowpea (Vigna unguiculata L.) leaves. To unravel early Mn-toxicity responses after 1-3 days Mn treatment also in the leaf symplast, we studied the symplastic reactions induced by Mn in two cultivars differing in Mn tolerance on a total cellular level. Comparative proteome analyses of plants exposed to low or high Mn allowed to identify proteins specifically affected by Mn, particularly in the Mn-sensitive cowpea cultivar. These proteins are involved in CO(2) fixation, stabilization of the Mn cluster of the photosystem II, pathogenesis-response reactions and protein degradation. Chloroplastic proteins important for CO(2) fixation and photosynthesis were of lower abundance upon Mn stress suggesting scavenging of metabolic energy for a specific stress response. Transcriptome analyses supported these findings, but additionally revealed an upregulation of genes involved in signal transduction only in the Mn-sensitive cultivar. In conclusion, a coordinated interplay of apoplastic and symplastic reactions seems to be important during the Mn-stress response in cowpea.

Heavy metals are important regulators of cell apoptosis. Manganese (Mn(2+)) is a potent inducer of apoptosis in different cell types, but the precise mechanisms that mediate such effects are not well defined. We previously reported that Mn(2+) was a potent apoptotic agent in human B cells, including lymphoma B cell lines. We show here that Mn(2+)-induced cell death in human B cells is associated with caspase-8-dependent mitochondrial activation leading to caspase-3 activity and apoptosis. We used specific caspase-8 interfering shRNAs to reduce caspase-8 expression, and this also reduced Mn(2+)-induced caspase-3 activation and apoptosis. Mn(2+)-triggered caspase-8 activation is associated with a specific pathway, which is independent of Fas-associated death domain protein, and dependent on the sequential activation of p38-mitogen-activated protein kinase (p38 MAPK) and mitogen- and stress-response kinase 1 (MSK1). Inhibition of p38 activity using either pharmacological inhibitors or dominant-negative mutant forms of p38 blocked Mn(2+)-mediated phosphorylation of MSK1 and blocked subsequent caspase-8 activation. However, specific inhibitors and the expression of a dominant-interfering mutant of MSK1 only inhibited caspase-8 activation, but not p38 activity. These findings suggest a novel model for the regulation of caspase-8 during Mn(2+)-induced apoptosis based on the sequential activation of p38 MAPK, MSK1, caspase-8 and mitochondria, respectively.

We have shown recently that Mn2+ stimulates gonadotropin secretion via an action at the hypothalamic level, and a diet supplemented with a low dose of the element is capable of advancing the time of female puberty. In this study, we used an in vitro approach to investigate the mechanism by which Mn2+ induces luteinizing hormone-releasing hormone (LHRH) secretion from prepubertal female rats. The medial basal hypothalamus from 30-day-old rats was incubated in Locke solution for 30 min to assess basal LHRH secretion, then incubated with buffer alone or buffer plus either a nitric oxide synthase (NOS) inhibitor (N-monomethyl-L-arginine (NMMA); 300 or 500 microM) or a soluble guanylyl cyclase (sGC) inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ); 100 or 250 microM) for another 30 min. Finally, the incubation continued for a further 30 min, but in the presence of MnCl2 (50 or 250 microM) to assess the effect of the blockers on stimulated LHRH secretion. Both 50 and 250 microM MnCl2 stimulated LHRH release (P < 0.05 and P < 0.01, respectively). The addition of 300-500 microM NMMA to the medium did not block Mn2+-stimulated release of LHRH, even with the higher dose of MnCl2. Furthermore, while 50, 100 and 250 microM MnCl2 all significantly induced LHRH release, the two lowest doses did not stimulate total nitrite released from the same tissue, an effect only observed with the highest dose. Taken together, these data suggest that Mn2+ is not an effective stimulator of NO. Conversely, inhibiting sGC with ODQ blocked the Mn2+-stimulated secretion of LHRH in a dose-dependent manner, indicating that GC is the site of action of Mn2+. Additionally, we showed that Mn2+ stimulated cGMP and LHRH from the same tissues, and that downstream blocking of protein kinase G formation with KT5823 (10 microM) inhibited Mn2+-induced LHRH release. These data demonstrate that the principal action of Mn2+ within the hypothalamus is to activate sGC directly and/or as a cofactor with available NO, hence generating cGMP and resulting in prepubertal LHRH release.

Macrophages are essential for the development of innate immune responses against a variety of infectious factors. They detect invading pathogens via their pattern recognition receptors such as toll-like receptors (TLRs). TLR7/8 recognizes ssRNA from various viruses. In the present study, we have used 2-DE gel-based proteomics to find novel TLR7/8 target proteins in human monocyte-derived macrophages in order to improve our understanding of the virus recognition by this TLR. A total of 27 protein spots were found to be reproducibly differentially expressed between control and TLR7/8 activated 2-DE gel pairs, 18 spots being more than two-fold upregulated and nine spots being at least two-fold downregulated. Several proteins involved in defense against toxic superoxide (O2-) and other reactive oxygen species, such as manganese-containing superoxide dismutase (SOD2), glutathione peroxidase, and peroxiredoxins 1 and 6 were highly upregulated after TLR7/8 activation. Western blot analysis showed that activation of macrophages with TLR2, TLR3, TLR4, and TLR7/8 ligands also strongly upregulated SOD2 protein expression. In conclusion, our results show that the activation of pattern recognition receptors of the innate immune system results in strong upregulation of SOD2 gene expression suggesting that SOD2 protects macrophages from oxidative stress during microbial infection.

Secretory-pathway Ca2(+)-transport ATPases (SPCA) provide the Golgi apparatus with Ca2+ and Mn2+ needed for the normal functioning of this organelle. Loss of one functional copy of the human SPCA1 gene (ATP2C1) causes Hailey-Hailey disease, a rare skin disorder characterized by recurrent blisters and erosions in the flexural areas. Here, we will review the properties and functional role of the SPCAs. The relationship between Hailey-Hailey disease and its defective gene (ATP2C1) will be adressed as well.

For enzymatic activities to be effectively carried out, basic prerequisites must be met. Many enzymatic tasks require continuous consumption and dissipation of energy, sometimes in massive amounts. Some activities, such as DNA replication, transcription, and repair through homologous recombination rely upon templates that provide the information required for these transactions. Yet, circumstances where intracellular energy pools are severely depleted, or where intact templates are not available, frequently occur. Moreover, the fact that in order to reach their targets, enzymes must cope with an extremely crowded and viscous cellular milieu that drastically slows down their diffusion is often neglected. These impediments are particularly evident under stress conditions such as prolonged starvation or continuous exposure to DNA-damaging agents. Here we survey recent studies, which imply that when enzymatically-mediated DNA repair pathways are hindered, alternative strategies are deployed, whose common denominator is the reorganization of bacterial nucleoids into morphologies that promote DNA repair and protection.

Friedreich ataxia is a human neurodegenerative and myocardial disease caused by decreased expression of the mitochondrial protein frataxin. Proteomic analysis of the mutant yeast model of Friedreich ataxia presented in this paper reveals that these cells display increased amounts of proteins involved in antioxidant defenses, including manganese-superoxide dismutase. This enzyme shows, however, lower activity than that found in wild type cells. Our results indicate that this lack of activity is a consequence of cellular manganese deficiency, because in manganese-supplemented cultures, cell manganese content, and manganese-superoxide dismutase activity were restored. One of the hallmarks of Friedreich ataxia is the decreased activity of iron/sulfur-containing enzymes. The activities of four enzymes of this group (aconitase, glutamate synthase, succinate dehydrogenase, and isopropylmalate dehydratase) have been analyzed for the effects of manganese supplementation. Enzyme activities were recovered by manganese treatment, except for aconitase, for which, a specific interaction with frataxin has been demonstrated previously. Similar results were obtained when cells were grown in iron-limited media suggesting that manganese-superoxide dismutase deficiency is a consequence of iron overload. In conclusion, these data indicate that generalized deficiency of iron-sulfur protein activity could be a consequence of manganese-superoxide dismutase deficiency, and consequently, it opens new strategies for Friedreich ataxia treatment.

Two areas of research have recently converged to highlight important roles for Mn(2+) in pathogenesis: the recognition that both bacterial Nramp homologs and members of LraI family of proteins are Mn(2+) transporters. Their mutation is associated with decreased virulence of various bacterial species. Thus, Mn(2+) appears to be essential for bacterial virulence. This review describes what is currently known about Mn(2+) transport in prokaryotes and how prokaryotic Mn(2+) transport is regulated. Some of the phenotypes that arise when microorganisms lack Mn(2+) are then discussed, with an emphasis on those phenotypes involving pathogenesis. The concluding section describes possible enzymatic roles for Mn(2+) that might help explain why Mn(2+) is necessary for virulence.

The perception and subsequent transduction of environmental signals are primary events in the acclimation of living organisms to changes in their environment. Many of the molecular sensors and transducers of environmental stress cannot be identified by traditional and conventional methods. Therefore, the genomic information has been exploited in a systematic approach to this problem, performing systematic mutagenesis of potential sensors and transducers, namely, histidine kinases and response regulators, respectively, in combination with DNA microarray analysis, to examine the genome-wide expression of genes in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Using targeted mutagenesis, 44 out of the 47 histidine kinases and 42 out of the 45 response regulators of this organism have successfully been inactivated. The resultant mutant libraries were screened by genome-wide DNA microarray analysis and by slot-blot hybridization analysis under various stress and non-stress conditions. Histidine kinases have been identified that perceive and transduce signals of low-temperature, hyperosmotic, and salt stress, as well as manganese deficiency.

Background and Aims: The objective of this research was to characterize the physiology and cell ultrastructure of two soybean genotypes subjected to nutrient solutions with increasing concentrations of manganese (Mn) at two contrasting iron (Fe) concentrations. Genotypes 'PI227557' and 'Biloxi' were selected based on their distinctly different capacities to accumulate Mn and Fe. *, Methods: Bradyrhizobium-inoculated plants were grown in hydroponic cultures in a greenhouse. Nutrient solutions were supplied with Mn concentrations ranging from 0.3 to 90 microm, at either 5 or 150 microm Fe as FeEDTA. *, Key Results: For both genotypes and at both Fe concentrations, Mn concentrations from 6.6 to 50 microm did not affect shoot, root and nodule mass, or leaf and nodule ureide concentration. Mn concentrations of 70 and 90 microm did not result in visible toxicity symptoms, but hindered growth and nodulation of 'Biloxi'. An Mn concentration of 0.3 microm was, however, deleterious to growth and nodulation for both genotypes, and caused an accumulation of ureides in leaves and major alterations in the ultrastructure of chloroplasts, nuclei and mitochondria, regardless of the Fe concentration. In 'PI227557', there was also a proliferation of Golgi apparatus and endoplasmic reticulum in the cytoplasm of leaf cells, and nodules showed disrupted symbiosomes lacking poly-beta-hydroxybutirate grains concomitantly with a proliferation of endoplasmic reticulum as well as arrested bacterial division. At 15 microm Fe, ferritin-like crystals were formed in the lumen of chloroplasts of 'PI227557' plants. For both genotypes, there was an antagonism between the Fe and Mn concentrations in leaves, the higher values of both microelements being detected in 'PI227557'. The absence of any detectable relationship between Fe or Mn and zinc, phosphorus and copper concentrations in leaves ruled out those micronutrients as relevant for Mn and Fe nutrition in soybeans. *, Conclusions: The results confirmed the greater capacity of 'PI227557' for Mn and Fe accumulation than 'Biloxi' for most nutrient treatments. Hence, 'PI227557' may be a very useful genetic resource both in developing soybean cultivars for growth on low nutrient soils and in physiological studies to understand differing approaches to nutrient accumulation in plants.

Deinococcus radiodurans is extremely resistant to ionizing radiation. How this bacterium can grow under chronic gamma radiation [50 grays (Gy) per hour] or recover from acute doses greater than 10 kGy is unknown. We show that D. radiodurans accumulates very high intracellular manganese and low iron levels compared with radiation-sensitive bacteria and that resistance exhibits a concentration-dependent response to manganous chloride [Mn(II)]. Among the most radiation-resistant bacterial groups reported, Deinococcus, Enterococcus, Lactobacillus, and cyanobacteria accumulate Mn(II). In contrast, Shewanella oneidensis and Pseudomonas putida have high iron but low intracellular manganese concentrations and are very sensitive. We propose that Mn(II) accumulation facilitates recovery from radiation injury.

Trace metals such as zinc, manganese, and iron are necessary for the growth and function of the brain. The transport of trace metals into the brain is strictly regulated by the brain barrier system, i.e., the blood-brain and blood-cerebrospinal fluid barriers. Trace metals usually serve the function of metalloproteins in neurons and glial cells, while a portion of trace metals exists in the presynaptic vesicles and may be released with neurotransmitters into the synaptic cleft. Zinc and manganese influence the concentration of neurotransmitters in the synaptic cleft, probably via the action against neurotransmitter receptors and transporters and ion channels. Zinc may be an inhibitory neuromodulator of glutamate release in the hippocampus, while neuromodulation by manganese might mean functional and toxic aspects in the synapse. Dietary zinc deficiency affects zinc homeostasis in the brain, followed by an enhanced susceptibility to the excitotoxicity of glutamate in the hippocampus. Transferrin may be involved in the physiological transport of iron and manganese into the brain and their utilization there. It is reported that the brain transferrin concentration is decreased in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease and that brain iron metabolism is also altered. The homeostasis of trace metals in the brain is important for brain function and also for the prevention of brain diseases.

Fe2+ has traditionally been considered the most important divalent cation involved in host-pathogen interactions. However, recent research indicates a previously unappreciated role for transition metal divalent cations other than Fe2+ during infection. Recent studies have identified an absolute requirement for Mn2+ in bacterial pathogens that are Fe2+-independent, indicating an important role for Mn2+ in pathogenesis. Potential roles for Mn2+ in pathogenesis include effects on the detoxification of reactive oxygen intermediates (ROIs), as a cofactor for enzymes involved in intermediary metabolism and signal transduction, and as a stimulus for virulence gene regulation. This review focuses on how these possible roles for Mn2+ may affect bacterial pathogenesis and the outcome of an infection.

During the past decade, numerous Mn2+-dependent protein serine, threonine and/or tyrosine phosphatases (O-phosphatases) from prokaryotes have been characterized. Based on their amino acid sequences, they belong to PPP, PPM or PHP superfamilies. Both the PPP and PPM families of protein phosphatases are metalloenzymes which active centers contain two metal ions that function as cofactors. Results from sequence analysis also suggest that PHP family protein phosphatase is a metalloenzyme. The identified functions for PPP family protein phosphatases from different prokaryotic organisms include regulation of stress-response, nitrogen fixation and vegetative growth. At least one phosphatase, PrpB from Escherichia coli, is also implicated in bacterial pathogenesis. Prokaryotic PPM family protein phosphatases are involved in controlling spore formation, stress-response, cell density during stationary phase, carbon and nitrogen assimilation, vegetative growth, development of fruiting bodies and cell segregation. The function of CpsB, a PHP family protein tyrosine phosphatase from Streptococcus pneumonia, is to regulate biosynthesis of capsular polysaccharide, an important virulence determinant. Thus, this group of functionally diverse protein phosphatases plays an important role in prokaryotes. Discovery of Mn2+-dependent prokaryotic protein O-phosphatases and their functions also contributes to new insight into Mn2+ homeostasis and many roles played by Mn2+ and protein O-phosphorylation in prokaryotic cells.

High-level occupational manganese (Mn) exposure has been reported to induce irreversible brain alterations determining a Parkinson-like disease. This study aimed to assess subclinical neurophysiological alterations in welding workers. They were employed in a machine building factory with an average Mn exposure <200 mg/m3. Sixty-eight workers (mean age: 34 years; mean Mn exposure duration: 16 years) and 42 flour factory workers (control group) with similar age and smoking habit were recruited. Autonomic nervous function test battery (ANSFT), composed of Valsalva maneuvre-induced heart rate variation (HR-V), heart rate variation following deep breathing (HR-DB) and heart rate variation following immediate standing up (HR-IS) was assessed. Electroencephalogram (EEG), brain electricity activity mapping (BEAM) were also performed. HR-V, HR-DB, and HR-IS were significantly lower in Mn-exposed subjects showing altered autonomic nervous system activity, parasympathetic-sympathetic imbalance and, and consequently, altered cardiovascular regulation and reactivity. The EEG of the Mn-exposed workers evidenced beta-wave rhythms significantly reduced, theta-waves markedly increased and abnormal wave activities of either localized or diffusive type. In the same workers BEAM revealed higher theta, delta and beta power values in the F7 area, lower d power values in the FP1, FP2 and C4 areas as well as dissymmetry in the central area, parietal region and occipital region. This study suggests that Mn impairs neuron activity within central nervous system. In this context, brainstem parasympathetic and sympathetic centers receiving axon projections from cortical and diencephalic areas, may reflect Mn effects on upper pathways. However, direct actions of Mn on these centers cannot be excluded.

Integrin alpha(4)beta(7) mediates rolling adhesion in Ca(2+) and Ca(2+) + Mg(2+), and firm adhesion in Mg(2+) and Mn(2+), mimicking the two key steps in leukocyte accumulation in inflamed vasculature. We mutated an interlinked linear array of three divalent cation-binding sites present in integrin beta-subunit I-like domains. The middle, metal ion-dependent adhesion site (MIDAS) is required for both rolling and firm adhesion. One polar site, that adjacent to MIDAS (ADMIDAS), is required for rolling because its mutation results in firm adhesion. The other polar site, the ligand-induced metal binding site (LIMBS), is required for firm adhesion because its mutation results in rolling. The LIMBS mediates the positive regulatory effects of low Ca(2+) concentrations, whereas the ADMIDAS mediates the negative regulatory effects of higher Ca(2+) concentrations, which are competed by Mn(2+). The bipolar sites thus stabilize two alternative phases of adhesion.

Chelating agents have been shown to induce characteristic changes in the light-minus-dark Fourier transform infrared (FTIR) difference spectrum for the S(2)/S(1) difference in the oxygen-evolving complex (OEC). Addition of various ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA)-type chelators, such as EDTA, O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid (EGTA), trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CyDTA), or N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA), to Ca(2+)-depleted PS II membranes resulted in the suppression of typical S(2)/S(1) vibrational features, including the symmetric (1365(+)/1404(-) cm(-1)) and the asymmetric (1587(+)/1566(-) cm(-1)) carboxylate stretching vibrations, as well as the amide I and II modes of the backbone polypeptides. In contrast, the addition of ethylenediamine-N,N'-diacetic acid (EDDA) showed less inhibitory effects. The effects of the chelators depended on the number of the carboxylate groups; chelators with more than two carboxymethyl groups were effective in altering the FTIR spectrum. The bridging structure that connects the two nitrogen atoms also influenced the inhibitory effects. However, the effects were not necessarily correlated with the stability constants of the chelators to Mn(2+). The vibrational modes that were suppressed by EDTA were almost completely restored by subsequent washing with Chelex-treated Ca(2+)-free buffer medium, indicating that the spectral changes are attributable to the reversible association of chelators with the Ca(2+)-depleted OEC. Nevertheless, prolonged incubation with chelators led to the impairment of the O(2)-evolving capability, with differences in the effectiveness, in the order that is consistent with that for the suppression effects on FTIR spectra. Chelators with carboxylate and/or carboxymethyl groups bound to a single nitrogen [nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA)] or carbon (citric acid) were relatively ineffective for the suppression. A chelator that includes four phosphate groups, ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic) acid (EDTPO), also showed suppression effects on both the carboxylate and amide modes. Based on these findings, a possible mode of interaction between the chelators and the Mn cluster is discussed.

The beta-diketo acids (DKAs) represent a major advance for anti-HIV-1 integrase drug development. We compared the inhibition of HIV-1 integrase by six DKA derivatives using the wild-type enzyme or the double-mutant F185K/C280S, which has been previously used for crystal structure determinations. With the wild-type enzyme, we found that DKAs could be classified into two groups: those similarly potent in the presence of magnesium and manganese and those potent in manganese and relatively ineffective in the presence of magnesium. Both the aromatic and the carboxylic or tetrazole functions of DKAs determined their metal selectivity. The F185K/C280S enzyme was markedly more active in the presence of manganese than magnesium. The F185K/C280S integrase was also relatively resistant to the same group of DKAs that were potent in the presence of magnesium with the wild-type enzyme. Resistance was caused by a synergistic effect from both the F185K and C280S mutations. Molecular modeling and docking suggested metal-dependent differences for binding of DKAs. Molecular modeling also indicated that the tetrazole or the azido groups of some derivatives could directly chelate magnesium or manganese in the integrase catalytic site. Together, these experiments suggest that DKAs recognize conformational differences between wild-type and the double-mutant HIV-1 integrase, because they chelate the magnesium or manganese in the enzyme active site and compete for DNA binding.

Root hair formation is induced by low pH in lettuce ( Lactuca sativa L. cv. Grand Rapids) seedlings cultured in mineral medium. The role of mineral concentrations in this phenomenon was investigated, especially for manganese. When lettuce seedlings were cultured in media that were deficient in calcium (Ca), manganese (Mn), boron (B) or molybdenum (Mo), morphological changes were induced in roots. Deficiency of other nutrients had little effect on root hair formation. Ca or B deficiency inhibited the growth of the main root and the formation of root hairs, regardless of pH. Mn or Mo deficiency increased root hair formation at pH 6 and suppressed main root growth slightly. In contrast, increasing the Mn concentration suppressed low-pH-induced root hair formation. The Mn content of roots grown at pH 4 was only about 15% of that at pH 6. In contrast, the Mo content of roots grown at low pH was about six times that of roots grown at neutral pH. These results suggest that root hair formation induced by low pH is at least partly mediated by decreased Mn uptake in root cells.

The identity of the physiological metal cofactor for human methionine aminopeptidase-2 (MetAP2) has not been established. To examine this question, we first investigated the effect of eight divalent metal ions, including Ca(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+), on recombinant human methionine aminopeptidase apoenzymes in releasing N-terminal methionine from three peptide substrates: MAS, MGAQFSKT, and (3)H-MASK(biotin)G. The activity of MetAP2 on either MAS or MGAQFSKT was enhanced 15-25-fold by Co(2+) or Mn(2+) metal ions in a broad concentration range (1-1000 microM). In the presence of reduced glutathione to mimic the cellular environment, Co(2+) and Mn(2+) were also the best stimulators (approximately 30-fold) for MetAP2 enzyme activity. To determine which metal ion is physiologically relevant, we then tested inhibition of intracellular MetAP2 with synthetic inhibitors selective for MetAP2 with different metal cofactors. A-310840 below 10 microM did not inhibit the activity of MetAP2-Mn(2+) but was very potent against MetAP2 with other metal ions including Co(2+), Fe(2+), Ni(2+), and Zn(2+) in the in vitro enzyme assays. In contrast, A-311263 inhibited MetAP2 with Mn(2+), as well as Co(2+), Fe(2+), Ni(2+), and Zn(2+). In cell culture assays, A-310840 did not inhibit intracellular MetAP2 enzyme activity and did not inhibit cell proliferation despite its ability to permeate and accumulate in cytosol, while A-311263 inhibited both intracellular MetAP2 and proliferation in a similar concentration range, indicating cellular MetAP2 is functioning as a manganese enzyme but not as a cobalt, zinc, iron, or nickel enzyme. We conclude that MetAP2 is a manganese enzyme and that therapeutic MetAP2 inhibitors should inhibit MetAP2-Mn(2+).

On the basis of the evidence that manganese may be released along with glutamate into the extracellular space in the hippocampus and amygdala, the release of manganese and its influence in the striatum was examined by using the in vivo microdialysis method in the present study. The release of 54Mn previously taken up by the striatum into the extracellular space was enhanced during stimulation with 100 mM KCl. This enhancement of 54Mn release into the striatal extracellular space was inhibited by addition of 1 micro M tetrodotoxin. When the rat striatum was perfused with artificial CSF containing 200 nM MnCl(2), the levels of GABA in the perfusate were remarkably decreased, while the levels of glutamate, aspartate, and glycine in the perfusate were not appreciably decreased. These results suggest that manganese released into the synaptic cleft in a calcium- and impulse-dependent manner may influence GABA release in the striatum.

There is increasing recognition of the emerging role of manganese regulation and acquisition in some pathogenic bacteria. Expression of the Enterococcus faecalis endocarditis-associated virulence factor EfaA is induced by growth in serum. It is demonstrated here that expression of the efaCBA operon encoding a putative ABC-type transporter is regulated by Mn(2+). Transcription of efaCBA and EfaA production were repressed in Mn(2+)-supplemented medium. A Mn(2+)-responsive transcriptional regulator, EfaR, sharing 27 % identity with the Corynebacterium diphtheriae diphtheria toxin repressor (DtxR), was identified. In the presence of Mn(2+), EfaR protein bound in vitro to the efaC promoter region. Analysis of the E. faecalis V583 genome revealed ten additional putative EfaR-binding sites, suggesting that manganese availability could have a broader regulatory role in infection. The results identify a new Mn(2+)-sensing regulator in enterococci that regulates the expression of a virulence factor implicated in enterococcal endocarditis.