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.

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.

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.

Recurrent reactive hypoglycemia due to clozapine-induced glucose intolerance: A case report.

Background: Although diabetes is one of the most common side effects of clozapine, a medication for the treatment of schizophrenia, to the best of our knowledge no study exists on clozapine-induced glucose intolerance or hypoglycemia in patients with schizophrenia. Case Presentation: We report a case of schizophrenia with repeated reactive hypoglycemia due to abnormal glucose tolerance during clozapine treatment. During clozapine administration in patients with schizophrenia, it is necessary to monitor physical and psychiatric symptoms due to reactive hypoglycemia and hyperglycemia. If abnormal glucose tolerance is a concern, it should be promptly detected using blood or oral glucose tolerance tests. Conclusion: Early intervention for impaired glucose tolerance may prevent clozapine discontinuation due to diabetes or hyperglycemia.

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.

A silicon transporter in rice.

Silicon is beneficial to plant growth and helps plants to overcome abiotic and biotic stresses by preventing lodging (falling over) and increasing resistance to pests and diseases, as well as other stresses. Silicon is essential for high and sustainable production of rice, but the molecular mechanism responsible for the uptake of silicon is unknown. Here we describe the Low silicon rice 1 (Lsi1) gene, which controls silicon accumulation in rice, a typical silicon-accumulating plant. This gene belongs to the aquaporin family and is constitutively expressed in the roots. Lsi1 is localized on the plasma membrane of the distal side of both exodermis and endodermis cells, where casparian strips are located. Suppression of Lsi1 expression resulted in reduced silicon uptake. Furthermore, expression of Lsi1 in Xenopus oocytes showed transport activity for silicon only. The identification of a silicon transporter provides both an insight into the silicon uptake system in plants, and a new strategy for producing crops with high resistance to multiple stresses by genetic modification of the root's silicon uptake capacity.

[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.

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.

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.

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.

Toward mechanistic elucidation of iron acquisition in barley: efficient synthesis of mugineic acids and their transport activities.

Iron acquisition of graminaceous plants is characterized by the synthesis and secretion of iron-chelating compounds, mugineic acids (MAs), and by a specific uptake system for MAs-iron(III) complexes. We identified a transporter, HvYS1 (Hordeum vulgare L. yellow stripe 1), that is highly specific for MAs-iron(III) in barley roots. In this article we outline the characterization of HvYS1, and our recent work on the practical syntheses of MAs and investigations into the molecular basis of the specific transport of their iron(III) complexes by HvYS1. 2'-Deoxymugineic acid (DMA) was synthesized in a good overall yield from commercially available Boc-l-allylglycine using a minimal number of short simple operations with minimal protecting groups and work-up/purification procedures. The same strategy was also successfully applied to beta-hydroxy-l-allylglycine, which was obtained by an allylic oxidation of l-allylglycine derivatives, to give MA and 2'-epi-MA efficiently. HvYS1 transported the iron(III) complexes of all three synthetic specimens with efficiency similar to that of a natural mugineic acid complex. With sufficient quantities of MAs in hand, we analyzed the function of HvYS1 and revealed by preparing chimeric transporters that the sixth outer membrane loop of the transporter plays a vital role in substrate specificity.

[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.

Structural element responsible for the Fe(III)-phytosiderophore specific transport by HvYS1 transporter in barley.

Hordeum vulgare L. yellow stripe 1 (HvYS1) is a selective transporter for Fe(III)-phytosiderophores, involved in primary iron acquisition from soils in barley roots. In contrast, Zea mays yellow stripe 1 (ZmYS1) in maize possesses broad substrate specificity, despite a high homology with HvYS1. Here we revealed, by assessing the transport activity of a series of HvYS1-ZmYS1 chimeras, that the outer membrane loop between the sixth and seventh transmembrane regions is essential for substrate specificity. Circular dichroism spectra indicated that a synthetic peptide corresponding to the loop of HvYS1 forms an alpha-helix in solution, whereas that of ZmYS1 is flexible. We propose that the structural difference at this particular loop determines the substrate specificity of the HvYS1 transporter.

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.

Antiganglion neuron antibodies correlate with neuropathy in Sjögren's syndrome.

To investigate the possible implication of antibodies against dorsal root ganglion neuron in the pathogenesis of sensory neuropathy with Sjögren's syndrome, we examined the pathogenic role of antiganglion neuron antibodies by immunoblotting, immunohistochemistry and immunoreactive assay. Sjögren's syndrome patients without neuropathy, patients with vasculitic neuropathy and normal volunteers were evaluated as controls. Antiganglion neuron antibodies recognizing certain proteins of several different molecular weights were detected only in patients of sensory neuropathy with Sjögren's syndrome. Those antibodies labeled specific-sized neurons in the fixed ganglion and isolated ganglion neurons under the culture condition, each of which corresponded well to clinical manifestations. These results suggest that antiganglion neuron antibodies may contribute to the pathogenesis of sensory neuropathy with Sjögren's syndrome.

[A case of stroke-like episode of MELAS of which progressive spread would be prevented by edaravone].

A thirty-two-year-old woman who had been diagnosed MELAS with 3243A > G mutation presented headache, nausea, decreased bilateral visual acuity, and topographical disturbance on January 1 in 2002. Although brain CT showed no fresh lesion, recurrence of stroke-like episode was considered. Immediately, she was treated with ubiquinone (210 mg/day, p.o.) and tocopherol nicotinate (300 mg/day, p.o.). She became confused on the fifth day. Diffusion weighted- and T2 weighted-MRI revealed appearance of hyperintense lesion at the right occipital lobe. We started edaravone infusion (30 mg, twice a day, div.) for two weeks with informed consent from her family. On 13th day her consciousness was improved. Edema and signal intensity of the lesion were decreased on MRI with minimal spread to the parietal lobe. She discharged on the 30th day with marked visual field loss, hemispatial neglect, and topographical amnesia. MRI after four months showed remarkable atrophy of the right occipital region. In our department, five stroke-like episodes including this case were treated with ubuiquinone and tocopherol nicotinate. This regimen was effective in prevention of progressive spread of lesions only in two episodes. Edaravone is radical scavenger used in acute cerebral infarction. Progressive spread into the neighboring regions is one of characteristics of MELAS, although its precise mechanisms are not well known. Oxidative stress induced by released free radicals through mitochondrial dysfunction might be one of factors and edaravone would make an effect through blockage of the free radicals. Edaravone could not rescue neurons in the initial lesion. Although more numbers of cases are needed to establish the effect of edaravone on MELAS, it could minimize the neurological deficits after stroke-like episode of MELAS.

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.