Epoxide Ring-Opening Reactions for Abundant Production of Mugineic Acids and Nicotianamine Probes.

A succinct synthetic approach to mugineic acids and 2'-hydroxynicotianamine was established. Unlike all other synthetic methods, this approach utilized epoxide ring-opening reactions to form two C-N bonds and is characterized by the absence of redox reactions. Mugineic acid was synthesized from three readily available fragments on a gram scale in 6 steps. The protected 2'-hydroxynicotianamine was also synthesized in 4 steps, and the dansyl group, serving as a fluorophore, was introduced through a click reaction after propargylation of the 2'-hydroxy group. The dansyl-labeled nicotianamine (NA) iron complexes were internalized by oocytes overexpressing ZmYS1 (from maize) or PAT1 (from human) transporters, indicating successful transport of the synthesized NA-probe through these transporters.

Uptake mechanism of iron-phytosiderophore from the soil based on the structure of yellow stripe transporter.

Calcareous soils cover one-third of all land and cause severe growth defects in plants due to the poor water solubility of iron at high pH. Poaceae species use a unique chelation strategy, whereby plants secrete a high-affinity metal chelator, known as phytosiderophores (mugineic acids), and reabsorb the iron-phytosiderophore complex by the yellow stripe 1/yellow stripe 1-like (YS1/YSL) transporter for efficient uptake of iron from the soil. Here, we present three cryo-electron microscopy structures of barley YS1 (HvYS1) in the apo state, in complex with an iron-phytosiderophore complex, Fe(III)-deoxymugineic acid (Fe(III)-DMA), and in complex with the iron-bound synthetic DMA analog (Fe(III)-PDMA). The structures reveal a homodimeric assembly mediated through an anti-parallel ß-sheet interaction with cholesterol hemisuccinate. Each protomer adopts an outward open conformation, and Fe(III)-DMA is bound near the extracellular space in the central cavity. Fe(III)-PDMA occupies the same binding site as Fe(III)-DMA, demonstrating that PDMA can function as a potent fertilizer in an essentially identical manner to DMA. Our results provide a structural framework for iron-phytosiderophore recognition and transport by YS1/YSL transporters, which will enable the rational design of new, high-potency fertilizers.

Development of a mugineic acid family phytosiderophore analog as an iron fertilizer.

Iron (Fe) is an essential nutrient, but is poorly bioavailable because of its low solubility in alkaline soils; this leads to reduced agricultural productivity. To overcome this problem, we first showed that the soil application of synthetic 2'-deoxymugineic acid, a natural phytosiderophore from the Poaceae, can recover Fe deficiency in rice grown in calcareous soil. However, the high cost and poor stability of synthetic 2'-deoxymugineic acid preclude its agricultural use. In this work, we develop a more stable and less expensive analog, proline-2'-deoxymugineic acid, and demonstrate its practical synthesis and transport of its Fe-chelated form across the plasma membrane by Fe(III)•2'-deoxymugineic acid transporters. Possibility of its use as an iron fertilizer on alkaline soils is supported by promotion of rice growth in a calcareous soil by soil application of metal free proline-2'-deoxymugineic acid.

Iron uptake mediated by the plant-derived chelator nicotianamine in the small intestine.

Iron is an essential metal for all living organisms that is absorbed in the intestinal cells as a heme-chelated or free form. It is unclear how important plant-derived chelators, such as nicotianamine (NA), an organic small molecule that is ubiquitous in crops, vegetables, and various other foods, contribute to iron bioavailability in mammals. We performed electrophysiological assays with Xenopus laevis oocytes and radioactive tracer experiments with Caco-2 cells. The findings revealed that the proton-coupled amino acid transporter SLC36A1 (PAT1) transports iron in the form of NA-Fe (II) complex in vitro. Decreased expression of hPAT1 by RNA interference in Caco-2 cells reduced the uptake of NA-59Fe (II) complex. The uptake of inorganic 59Fe (II) was relatively unaffected. These results imply that PAT1 transports iron as a NA-Fe (II) complex. The rate of 59Fe absorption in the spleen, liver, and kidney was higher when mice were orally administered NA-59Fe (II) compared with free 59Fe (II). The profile of site-specific PAT1 expression in the mouse intestine coincided with those of NA and iron contents, which were the highest in the proximal jejunum. Orally administered NA-59Fe (II) complex in mice was detected in the proximal jejunum by thin layer chromatography. In contrast, much less 59Fe (or NA) was detected in the duodenum, where the divalent metal transporter SLC11A2 (DMT1) absorbs free Fe (II). The collective results revealed the role of PAT1 in NA-Fe (II) absorption in the intestine and potential implication of NA in iron uptake in mammals.

Organic Chemistry Research on the Mechanistic Elucidation of Iron Acquisition in Barley.

An organic chemistry approach to the mechanistic elucidation of iron acquisition in graminaceous plants is introduced here. To elucidate this detailed mechanism using phytosiderophores, the efficient synthesis of 2'-deoxymugineic acid (DMA), a phytosiderophore of rice, was established. The synthetic DMA was confirmed to have similar iron transport activity to that of natural mugineic acid (MA). It was also revealed that the addition of synthetic DMA, along with iron, to a rice hydroponic solution enabled the rice to grow well even under an alkaline condition, and DMA clearly showed its high potential as a fertilizer to improve food production. On the other hand, the 2'-hydroxy group of MA was confirmed to serve as a point of introduction for labeling, allowing the synthesis of various mugineic acid derivatives as molecular probes. The incorporation of fluorescent mugineic acid into cells allowed them to be clearly observed by fluorescence confocal analysis, and this provided the first direct experimental evidence of transporter-mediated internalization of mugineic acid into cells.

The helical propensity of the extracellular loop is responsible for the substrate specificity of Fe(III)-phytosiderophore transporters.

Hordeum vulgare L. yellow stripe 1 (HvYS1) is a selective transporter of Fe(III)-phytosiderophores in barley that is responsible for iron acquisition from the soil. In contrast, maize Zea mays, yellow stripe 1 (ZmYS1) possesses broad substrate specificity. In this study, a quantitative evaluation of the transport activities of HvYS1 and ZmYS1 chimera proteins revealed that the seventh extracellular membrane loop is essential for substrate specificity. The loop peptides of both transporters were prepared and analysed by circular dichroism and NMR. The spectra revealed a higher propensity for α-helical conformation of the HvYS1 loop peptide and a largely disordered structure for that of ZmYS1. These structural differences are potentially responsible for the substrate specificities of the transporters.

A case of autoimmune epilepsy associated with anti-leucine-rich glioma inactivated subunit 1 antibodies manifesting electrical shock-like sensations and transparent sadness.

Autoimmune epilepsy is an isolated phenotype of autoimmune encephalitis, which may be suspected in patients with unexplained adult-onset seizure disorders or resistance to antiepileptic drugs (AEDs). Antibodies against leucine-rich glioma inactivated subunit 1 of the voltage-gated potassium channel (VGKC) complex, recently termed anti-LGI-1 antibodies, are one of the causes of autoimmune epilepsies. Bizarre symptoms with extremely short duration and high frequency are clues to the possible presence of autoimmune epilepsy with anti-LGI-1 antibodies. Precise diagnosis is important because autoimmune epilepsy is treatable and the prognosis can be predicted.

Phytosiderophores revisited: 2'-deoxymugineic acid-mediated iron uptake triggers nitrogen assimilation in rice (Oryza sativa L.) seedlings.

Poaceae plants release phytosiderophores into the rhizosphere in order to chelate iron (Fe), which often exists in insoluble forms especially under high pH conditions. The impact of phytosiderophore treatment at the physiological and molecular levels in vivo remains largely elusive, although the biosynthesis of phytosiderophores and the transport of phytosiderophore-metal complexes have been well studied. We recently showed that the application of 30 µM of the chemically synthesized phytosiderophore 2'-deoxymugineic acid (DMA) was sufficient for apparent full recovery of otherwise considerably reduced growth of hydroponic rice seedlings at high pH. Moreover, unexpected induction of high-affinity nitrate transporter gene expression as well as nitrate reductase activity indicates that the nitrate response is linked to Fe homeostasis. These data shed light on the biological relevance of DMA not simply as a Fe chelator, but also as a trigger that promotes plant growth by reinforcing nitrate assimilation.

Evaluation of amygdala pathology using (11)C-methionine positron emission tomography/computed tomography in patients with temporal lobe epilepsy and amygdala enlargement.

OBJECTIVE: The association between amygdala enlargement (AE) and temporal lobe epilepsy (TLE) has increasingly been reported. However, the pathology of AE remains poorly understood. The purpose of this study was to explore AE pathology using (11)C-methionine (Met) positron emission tomography (PET)/computed tomography (CT) in patients who have TLE with AE. MATERIALS AND METHODS: Twenty-six TLE patients with AE and 18 TLE patients without AE underwent (11)C-Met PET/CT and magnetic resonance imaging (MRI). (11)C-Met uptake in amygdala was evaluated by both visual inspection and semi-quantitative measurement. MRI was assessed visually and semi-quantitatively for AE. Laterality index (LI) was obtained by comparing the amygdala volumes of ipsilateral and contralateral sides. The clinical course and histopathological findings of all patients were also analyzed. RESULTS: On (11)C-Met PET/CT images, visual examination detected increased uptake in the enlarged amygdala in 7 of 26 (27%) TLE patients with AE, and the results were confirmed by semi-quantitative analysis. Among six TLE patients with AE who underwent surgery, histopathology revealed neoplasms (low grade astrocytoma and gangliocytoma) in two patients and focal cortical dysplasia in one patient with increased (11)C-Met uptake, but non-neoplastic lesions (focal cortical dysplasia, vacuolar degeneration, and hamartoma) in all three patients with no increased (11)C-Met uptake. On MRI, volume of the affected amygdala was significantly larger compared to the contralateral amygdala. LI was significantly higher in the group with AE than in the group without AE. CONCLUSIONS: This study revealed that some TLE patients with AE showed increased (11)C-Met uptake in the enlarged amygdala. (11)C-Met PET/CT is potentially useful for the evaluation of AE pathology, and may provide beneficial information for appropriate decision-making.

Transgenic petunia with the iron(III)-phytosiderophore transporter gene acquires tolerance to iron deficiency in alkaline environments.

Iron is an essential nutrient for all plants. However, terrestrial plants often suffer from iron deficiency in alkaline soil due to its extremely low solubility. Alkaline soil accounts for about 30% of all cultivated ground in the world. Plants have evolved two distinct strategies, I and II, for iron uptake from the soil. Dicots and non-graminaceous monocots use Strategy I, which is primarily based on the reduction of iron(III) to iron(II) and the uptake of iron(II) by the iron-regulated transporter, IRT1. In contrast, graminaceous plants use Strategy II to efficiently acquire insoluble iron(III). Strategy II comprises the synthesis and secretion of iron-chelating phytosiderophores, such as mugineic acids and the Yellow Stripe 1 transporter proteins of the iron(III)-phytosiderophore complex. Barley, which exhibits the highest tolerance to iron deficiency in alkaline soil among graminaceous plants, utilizes mugineic acids and the specific iron(III)-mugineic acids transporter, HvYS1. In this study, we established the transgenic plant Petunia hybrida, which originally had only Strategy I, by introducing the HvYS1 transporter gene derived from barley. When the transgenic plants were grown hydroponically in media containing the iron(III)-2'-deoxymugineic acid complex, free 2'-deoxymugineic acid and its iron(III) complex were detected in the root extract of the transgenic plant by electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry. The growth of the transgenic petunia was significantly better than that of the control host in alkaline conditions. Consequently, the transgenic plant acquired a significantly enhanced tolerance to alkaline hydroponic media in the presence of the iron(III)-2'-deoxymugineic acid complex. Furthermore, the flower color of the transgenic plant deepened. The results showed that iron-phytosiderophore complexes and their transporters can potentially be utilized to overcome the worldwide iron uptake problems to diverse plant species that are found in areas with alkaline conditions.

Nonconvulsive status epilepticus in the elderly associated with newer antidepressants used at therapeutic doses: A report of three cases.

Classic antidepressants have been known to induce convulsive seizures and nonconvulsive status epilepticus (NCSE). On the other hand, many reports have emphasized the safety of novel antidepressants. However, we encountered three cases of NCSE in the elderly associated with the use of newer antidepressants at therapeutic doses. All three patients were male and were 73 years of age or older. One patient was recently diagnosed with temporal lobe epilepsy and treated with low-dose lamotrigine. In all patients, newer antidepressants were initiated because of depressive symptoms. After titrating to therapeutic doses (paroxetine 20 mg/day, sertraline 50 mg/day, and combination of sertraline 50 mg/day and mirtazapine 30 mg/day in one patient each), impaired consciousness appeared. Electroencephalography (EEG) showed generalized slow waves with intermittent spike-slow-wave complexes. Intravenous injection of antiepileptic drugs improved EEG findings and clinical symptoms. After discontinuance of the abovementioned antidepressants, NCSE did not recur in any of patients. These reports raise the question of whether the newer antidepressants, like classic antidepressants, might also induce NCSE in the elderly, even when used at therapeutic doses. Physicians should consider monitoring for possible NCSE when using newer antidepressants in patients who may have low drug tolerability. Active continuous video-EEG monitoring is essential when behavioral and psychological symptoms or change in consciousness level is suspected.

2'-Deoxymugineic acid promotes growth of rice (Oryza sativa L.) by orchestrating iron and nitrate uptake processes under high pH conditions.

Poaceae plants release 2'-deoxymugineic acid (DMA) and related phytosiderophores to chelate iron (Fe), which often exists as insoluble Fe(III) in the rhizosphere, especially under high pH conditions. Although the molecular mechanisms behind the biosynthesis and secretion of DMA have been studied extensively, little information is known about whether DMA has biological roles other than chelating Fe in vivo. Here, we demonstrate that hydroponic cultures of rice (Oryza sativa) seedlings show almost complete restoration in shoot height and soil-plant analysis development (SPAD) values after treatment with 3-30 µm DMA at high pH (pH 8.0), compared with untreated control seedlings at normal pH (pH 5.8). These changes were accompanied by selective accumulation of Fe over other metals. While this enhanced growth was evident under high pH conditions, DMA application also enhanced seedling growth under normal pH conditions in which Fe was fairly accessible. Microarray and qRT-PCR analyses revealed that exogenous DMA application attenuated the increased expression levels of various genes related to Fe transport and accumulation. Surprisingly, despite the preferential utilization of ammonium over nitrate as a nitrogen source by rice, DMA application also increased nitrate reductase activity and the expression of genes encoding high-affinity nitrate transporters and nitrate reductases, all of which were otherwise considerably lower under high pH conditions. These data suggest that exogenous DMA not only plays an important role in facilitating the uptake of environmental Fe, but also orchestrates Fe and nitrate assimilation for optimal growth under high pH conditions.

Chronic periodic lateralised epileptic discharges and anti-N-methyl-D-aspartate receptor antibodies.

Periodic lateralised epileptiform discharges (PLEDs) are uncommon transient electroencephalographic findings accompanied by acute brain lesions. A small proportion of PLEDs persist for more than three months and are called "chronic" PLEDs, the pathophysiology of which is still debated. Herein, we report a man with right hemispheric PLEDs which lasted for more than 14 months and mild left hemispatial neglect after he experienced status epilepticus. Although MRI was normal, positron emission tomography revealed right temporo-parieto-occipital hypometabolism, which coincided with the source area of PLEDs estimated by magnetoencephalography. In addition, levels of anti-N-methyl-D-aspartate (NMDA) receptor antibodies and granzyme B were found to be high in the cerebrospinal fluid. Following two courses of steroid pulse therapy, the patient's left spatial neglect improved and the PLEDs were partially resolved. These findings suggest that the chronic PLEDs present in this case were an interictal phenomenon and that their pathophysiology involved autoimmune processes.

A case of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) coexisting with pervasive developmental disorder harboring SCN1A mutation in addition to CHRNB2 mutation.

We report a case of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) with several characteristics distinct from previously reported cases, in which genetic studies identified mutations in two different genes. This case differed from typical ADNFLE with respect to the following: (1) slightly younger onset and refractory to antiepileptic drugs and (2) borderline intellectual functioning and coexistence of pervasive developmental disorder from infancy. Genetic testing revealed a novel mutation and a silent substitution in SCN1A (c.4285G>T, A1429S and c.4371G>C, silent) in addition to a known mutation in CHRNB2 (c.1200C>G, I312M). SCN1A is a gene that codes for the voltage-dependent sodium channel α1 subunit and has been implicated in generalized epilepsy with febrile seizures plus and severe myoclonic epilepsy in infancy. However, the relation between SCN1A and ADNFLE is unknown. We report the clinical course and symptomatic characteristics of this case although the relationship between ADNFLE mutation and SCN1A mutation remains to be elucidated.

[Report of a study concerning carry-over in epilepsy patients: questionnaire survey of neurologists].

We conducted a questionnaire survey on 7,500 members of the Societas Neurologica Japonica regarding the carry-over from pediatric to adult epilepsy care. The response rate was 16.9%. Analysis of the responses showed that 46% of the neurologists were aware of the term "carry-over" in epilepsy care, and 78% felt difficulties with epilepsy care in general. The most common reasons included "not familiar with interpreting EEG" and "not familiar with the laws, regulations and medical and welfare systems specific to epilepsy". Among the neurologists who felt difficulties when accepting epilepsy patients aged 20 years or older referred from the pediatric department, 68% had experienced accepting these patients. The two major reasons for feeling difficulties when accepting these patients were "difficult to have good understanding of the disease course from infancy" and "not familiar with the epilepsy syndrome specific to infancy". The above findings highlight the importance of recognizing the existence of the issue of carry-over in adult epilepsy care and its significance, and to resolve the factors that hinder the transition of care. To achieve this goal, joint activities of the Japanese Society of Child Neurology and the Japan Epilepsy Society in collaboration with the Societas Neurologica Japonica, the Japanese Society of Psychiatry and Neurology, and the Japan Neurosurgical Society are critical.

[Report of study on transitional medicine for epilepsy: questionnaire survey of pediatric neurologists].

We conducted a questionnaire survey to investigate the situation of transitional medicine for epilepsy. Among the epilepsy patients cared by pediatric neurologists, 27% were adult patients including some elderly epilepsy patients. Seventy-six percent of the pediatric neurologists felt some difficulties in providing care for adult epilepsy. The main issues were psychiatric/psychological co-morbidity, medical co-morbidity, and a lack of in-patient facilities in the vicinity. This survey demonstrated that the lack of the transitional medicine for epilepsy in Japan is a profound problem, and the factors that hamper transfer of epilepsy patients to adult specialties should be resolved. To solve this problem, it is imperative that the Japanese Society of Child Neurology and Japan Epilepsy Society conduct collaborative activities with three relevant societies; Societas Neurologica Japonica, the Japanese Society of Psychiatry and Neurology, and the Japan Neurosurgical Society.

A novel barley yellow stripe 1-like transporter (HvYSL2) localized to the root endodermis transports metal-phytosiderophore complexes.

Recent advances in our understanding of how graminaceous plants take up insoluble forms of iron from the rhizosphere and mobilize them in plant tissues are primarily based on the identification of various transporters that are specific to metal-phytosiderophore (PS) complexes containing mugineic acid and deoxymugineic acid. Barley (Hordeum vulgare L.) yellow stripe 1 (HvYS1) is a metal-PS transporter that preferentially transports Fe(III)-PS compared with other metal complexes. Here, we report the cloning and characterization of HvYSL2, a novel metal-PS transporter encoding gene. HvYSL2 is composed of 702 amino acids with 14 transmembrane domains, which are conserved among this class of transporters, and exhibits 67.3% identity to HvYS1. Electrophysiological experiments with Xenopus laevis oocytes revealed that HvYSL2 transports PS complexes with Fe(III), Zn(II), Ni(II), Cu(II), Mn(II) or Co(II); this constitutes a broader range of substrate preference than HvYS1. Real-time PCR analysis revealed that HvYSL2 mRNA is expressed in shoots and also in roots, where it is induced under iron-deficient conditions. Moreover, immunohistochemistry in roots revealed that HvYSL2 is localized to the endodermis, whereas HvYS1 is expressed primarily in the epidermis. These data suggest that HvYSL2 is spatially distinct from HvYS1 and plays a unique role in delivering a broad range of essential metals in barley.

[Monthly low-dose immunoglobulin infusion as a maintenance therapy for multifocal motor neuropathy may reduce allergic adverse effects: a case report].

A 41-year-old man with multiple motor neuropathy developed weakness of the left hand at the age of 35 years. The weakness gradually progressed to his right hand. High-dose intravenous immunoglobulin (IVIg) therapy (0.4 g/kg for 5 consecutive days) improved the muscle weakness in the hands but led to the development of generalized severe pompholyx that spread to the skin over the entire body. Because muscle weakness of the hands worsened several months after IVIg therapy, we attempted another course of IVIg therapy. However, antiallergic agents and oral corticosteroids did not suppress the pompholyx induced by the high-dose IVIg. Hence, the treatment was switched to low-dose immunoglobulin therapy (0.4 g/kg for one day) once every month. After more than 8 months of low-dose therapy, only mild form of pompholyx remained and the muscle strength was maintained without further deterioration.