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Focal epilepsy is associated with intermittent brief population discharges (interictal spikes), which resemble sentinel spikes that often occur at the onset of seizures. Why interictal spikes self-terminate whilst seizures persist and propagate is incompletely understood. We used fluorescent glutamate and GABA sensors in an awake rodent model of neocortical seizures to resolve the spatiotemporal evolution of both neurotransmitters in the extracellular space. Interictal spikes were accompanied by brief glutamate transients which were maximal at the initiation site and rapidly propagated centrifugally. GABA transients lasted longer than glutamate transients and were maximal ∼1.5 mm from the focus where they propagated centripetally. Prior to seizure initiation GABA transients were attenuated, whilst glutamate transients increased, consistent with a progressive failure of local inhibitory restraint. As seizures increased in frequency, there was a gradual increase in the spatial extent of spike-associated glutamate transients associated with interictal spikes. Neurotransmitter imaging thus reveals a progressive collapse of an annulus of feed-forward GABA release, allowing seizures to escape from local inhibitory restraint., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

This study investigated the effectiveness and safety of low-dose salvage craniospinal irradiation (CSI) for recurrent germinoma. We retrospectively reviewed long-term tumor control and late adverse effects in 15 recurrent germinoma patients treated at our hospital between 1983 and 2019. Following the first recurrence of germinoma, seven were treated with 24-30 Gy of salvage CSI, three underwent non-CSI, and five were treated with only chemotherapy. CSI achieved a significantly better recurrence-free survival rate after the first recurrence compared to other strategies (100% vs 33%, p < 0.001: log-rank test). To evaluate the safety of salvage CSI, we assessed the outcomes at the final follow-up of seven patients who received salvage CSI at first recurrence and three patients who received salvage CSI at second recurrence. The median follow-up period was 220 months after initial treatment. Five patients who received 40-50 Gy of radiation therapy or underwent multiple radiation therapy before salvage CSI were classified into Group A, whereas five patients treated with platinum-based chemotherapy and 24-32 Gy of radiation therapy to the primary site, whole ventricle, or whole brain were classified into Group B. In Group A, one had endocrine dysfunction and the other had visual dysfunction. None were socially independent. Meanwhile, in Group B, no endocrine or visual dysfunction was found, and three patients were socially independent. Salvage CSI achieved excellent tumor control in recurrent germinoma and was safe in patients initially treated with low-dose radiation therapy and chemotherapy., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

Non-convulsive status epilepticus describes the syndrome of unexplained impaired consciousness in critically ill patients. Non-convulsive status epilepticus is very likely to lead to delayed diagnosis and poor outcomes because of the absence of convulsive symptoms. EEG is essential for the diagnosis of non-convulsive status epilepticus to establish the association between periodic discharges and rhythmic delta activity in addition to ictal epileptiform discharges according to the Salzburg criteria. Arterial spin labelling, a type of perfusion MRI, has been applied for rapid and non-invasive evaluation of the ictal state. Ictal cerebral cortical hyperperfusion is the most common finding to demonstrate focal onset seizures. Hyperperfusion of the thalamus on single photon emission computed tomography was found in patients with impaired awareness seizures. We hypothesized that thalamocortical hyperperfusion on arterial spin labelling identifies non-convulsive status epilepticus and such thalamic hyperperfusion specifically associates with periodic/rhythmic discharges producing impaired consciousness without convulsion. We identified 27 patients (17 females; age, 39-91 years) who underwent both arterial spin labelling and EEG within 24 h of suspected non-convulsive status epilepticus. We analysed 28 episodes of suspected non-convulsive status epilepticus and compared hyperperfusion on arterial spin labelling with periodic/rhythmic discharges. We evaluated 21 episodes as a positive diagnosis of non-convulsive status epilepticus according to the Salzburg criteria. We identified periodic discharges in 15 (12 lateralized and 3 bilateral independent) episodes and rhythmic delta activity in 13 (10 lateralized, 1 bilateral independent and 2 generalized) episodes. Arterial spin labelling showed thalamic hyperperfusion in 16 (11 unilateral and 5 bilateral) episodes and cerebral cortical hyperperfusion in 24 (20 unilateral and 4 bilateral) episodes. Thalamic hyperperfusion was significantly associated with non-convulsive status epilepticus ( P  =   0.0007; sensitivity, 76.2%; specificity, 100%), periodic discharges ( P  <   0.0001; 93.3%; 84.6%), and rhythmic delta activity ( P  =   0.0006; 92.3%; 73.3%). Cerebral cortical hyperperfusion was significantly associated with non-convulsive status epilepticus ( P  =   0.0017; 100%; 57.1%) and periodic discharges ( P  =   0.0349; 100%; 30.8%), but not with rhythmic delta activity. Thalamocortical hyperperfusion could be a new biomarker of non-convulsive status epilepticus according to the Salzburg criteria in critically ill patients. Specific thalamic hyperexcitability might modulate the periodic discharges and rhythmic delta activity associated with non-convulsive status epilepticus. Impaired consciousness without convulsions could be caused by predominant thalamic hyperperfusion together with cortical hyperperfusion but without ictal epileptiform discharges., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain.)

We report a unique case of a Japanese woman with herpetiform pemphigus (HP) who had IgG autoantibodies reactive with nondesmosomal sites of keratinocytes and presented characteristic transmission electron microscopic (TEM) findings of various-sized vacuoles in keratinocytes without acantholysis. The patient presented with pruritic annular oedematous erythemas with small blisters lining the margins on the trunk and extremities. Histopathological examinations showed intraepidermal blisters with prominent infiltrations of eosinophils. Direct and indirect immunofluorescence tests revealed the presence of in vivo bound and circulating IgG autoantibodies to the keratinocyte cell surfaces. However, enzyme-linked immunosorbent assays for desmoglein (Dsg) 1, Dsg3 and desmocollins 1-3 showed negative results. Immunoblotting using the full-length human Dsg1 recombinant protein showed a positive band. TEM examination showed various-sized vacuoles squashing the nuclei in many keratinocytes, resulting in rupture of the cells. Immunoelectron microscopic examination revealed IgG deposition over the entire keratinocyte cell surfaces, which spared the desmosomes. IgG antibodies were also present on the inside walls of the vacuoles around the nuclei of keratinocytes and on the cell surfaces of infiltrating eosinophils. This patient also had marked eosinophilia and high levels of thymus and activation-regulated chemokine and interleukin-5 in the serum. These results indicated a novel autoantigen on the nondesmosomal keratinocyte cell surfaces and the pathogenesis of bullous spongiotic change with inflammation in HP., (© 2018 British Association of Dermatologists.)

Background: Although a number of pathological processes resulting in amyloid deposition have been described in lichen amyloidosus (LA), no attention has been paid to the involvement of sweat glands/ducts in the pathogenesis of LA. According to recent studies, follicular structures are usually spared in serial histological sections of LA, and deposits of amyloid are likely to be confined to areas that display xerosis, suggesting that decreases in skin wetness by sweating disturbance seem to initiate LA., Objectives: To investigate whether sweating disturbance could represent an early event that triggers LA, and whether resolution of LA could be induced by restoring the sweating disturbance., Methods: By using the impression mould technique, which allows an accurate quantification of individual sweat glands/ducts actively delivering sweat, we examined sweat responses to thermal stimulus in LA lesions before and after treatment with a moisturizer., Results: Sweating disturbance was most profoundly detected in the 'hub' structure of the LA papule, and this disturbance due to leakage of sweat could be restored by short-term treatment with a moisturizer, particularly when used under occlusion., Conclusions: This study was limited by the relatively small sample size. Treatment of LA should be primarily directed at preventing leakage of sweat into the dermis or epidermis and therefore sweat delivery to the skin surface could be made easier., (© 2016 British Association of Dermatologists.)

Pheophytin a is an essential component of oxygenic photosynthetic organisms because the primary charge separation between chlorophyll a and pheophytin a is the first step in the conversion of light energy. In addition, conversion of chlorophyll a to pheophytin a is the first step of chlorophyll degradation. Pheophytin is synthesized by extracting magnesium (Mg) from chlorophyll; the enzyme Mg-dechelatase catalyzes this reaction. In this study, we report that Mendel's green cotyledon gene, STAY-GREEN ( SGR ), encodes Mg-dechelatase. The Arabidopsis thaliana genome has three SGR genes, SGR1 , SGR2 , and STAY-GREEN LIKE ( SGRL ). Recombinant SGR1/2 extracted Mg from chlorophyll a but had very low or no activity against chlorophyllide a ; by contrast, SGRL had higher dechelating activity against chlorophyllide a compared with chlorophyll a All SGRs could not extract Mg from chlorophyll b Enzymatic experiments using the photosystem and light-harvesting complexes showed that SGR extracts Mg not only from free chlorophyll but also from chlorophyll in the chlorophyll-protein complexes. Furthermore, most of the chlorophyll and chlorophyll binding proteins disappeared when SGR was transiently expressed by a chemical induction system. Thus, SGR is not only involved in chlorophyll degradation but also contributes to photosystem degradation., (© 2016 American Society of Plant Biologists. All rights reserved.)

Tctex1d2 (Tctex1 domain containing 2) is an open reading frame that encodes for a functionally unknown protein that contains a Tctex1 domain found in dynein light chain family members. Examination of gene expression during adipogenesis demonstrated a marked increase in Tctex1d2 protein expression that was essentially undetectable in preadipocytes and markedly induced during 3T3-L1 adipocyte differentiation. Tctex1d2 overexpression significantly inhibited insulin-stimulated glucose transporter 4 (GLUT4) translocation and 2-deoxyglucose uptake. In contrast, Tctex1d2 knockdown significantly increased insulin-stimulated GLUT4 translocation and 2-deoxyglucose uptake. However, acute insulin stimulation (up to 30 min) in 3T3-L1 adipocytes with overexpression or knockdown of Tctex1d2 had no effect on Akt phosphorylation, a critical signal transduction target required for GLUT4 translocation. Although overexpression of Tctex1d2 had no significant effect on GLUT4 internalization, Tctex1d2 was found to associate with syntaxin 4 in an insulin-dependent manner and inhibit Doc2b binding to syntaxin 4. In addition, glucose-dependent insulinotropic polypeptide rescued the Tctex1d2 inhibition of insulin-stimulated GLUT4 translocation by suppressing the Tctex1d2-syntaxin 4 interaction and increasing Doc2b-Synatxin4 interactions. Taking these results together, we hypothesized that Tctex1d2 is a novel syntaxin 4 binding protein that functions as a negative regulator of GLUT4 plasma membrane translocation through inhibition of the Doc2b-syntaxin 4 interaction.

Legumes produce root nodules as symbiotic organs where nitrogen-fixing bacteria are accommodated. Lotus japonicus NODULE INCEPTION (NIN) is an essential factor that specifically and positively regulates nodulation processes, and has evolved from a member of the NIN-like proteins, of which Arabidopsis homologs target nitrate-responsive elements (NREs), and activate gene expression in response to nitrate. It is therefore assumed that the NIN-mediated transcriptional network overlaps with those regulated by NLPs, because of their common DNA-binding RWP-RK domains. However, nodulation is inhibited in the presence of nitrate, and involvement of NIN in nitrate responses has remained largely unknown. Here we determined a consensus of NIN-binding nucleotide sequences (NBSs) by in vitro experiments, and revealed that the sequence pattern was very similar to those of NREs. Chromatin immunoprecitiation (ChIP)-PCR analyses showed that NIN targeted NREs in L. japonicus nitrate-inducible gene promoters, including LjNIR1, LjNRT2.1 and LjNRT2.2. Affinities of NIN binding to the NREs were comparable with that to NBS-yB1a, an NBS on the symbiotic LjNF-YB1 promoter, indicating that NREs are potential targets of NIN. However, rhizobial infection did not activate LjNIR1, LjNRT2.1 and LjNRT2.2. NIN ectopic expression interfered with nitrate-dependent activation of these genes. Nitrate treatment followed by NIN activation down-regulated expression of symbiotic NIN target genes. Our results showed that NIN and nitrate antagonistically regulate expression of genes that are activated by nitrate and NIN, respectively. We propose that this antagonistic relationship prevents inappropriate activation of genes in response to nitrate and rhizobial infection., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Ca(2+)/calmodulin (CaM)-dependent protein kinase (CCaMK) is a key regulator of root nodule and arbuscular mycorrhizal symbioses and is believed to be a decoder for Ca(2+) signals induced by microbial symbionts. However, it is unclear how CCaMK is activated by these microbes. Here, we investigated in vivo activation of CCaMK in symbiotic signaling, focusing mainly on the significance of and epistatic relationships among functional domains of CCaMK. Loss-of-function mutations in EF-hand motifs revealed the critical importance of the third EF hand for CCaMK activation to promote infection of endosymbionts. However, a gain-of-function mutation (T265D) in the kinase domain compensated for these loss-of-function mutations in the EF hands. Mutation of the CaM binding domain abolished CaM binding and suppressed CCaMK(T265D) activity in rhizobial infection, but not in mycorrhization, indicating that the requirement for CaM binding to CCaMK differs between root nodule and arbuscular mycorrhizal symbioses. Homology modeling and mutagenesis studies showed that the hydrogen bond network including Thr265 has an important role in the regulation of CCaMK. Based on these genetic, biochemical, and structural studies, we propose an activation mechanism of CCaMK in which root nodule and arbuscular mycorrhizal symbioses are distinguished by differential regulation of CCaMK by CaM binding.

Lipopolysaccharide (LPS) is a bacterial molecule that induces nitric oxide (NO) production and triggers defense systems in plant-pathogen interactions. NO production is induced in the roots of Lotus japonicus after inoculation of the roots with its microsymbiont Mesorhizobium loti. However, the rhizobial molecule that induces NO production has not yet been identified. We investigated NO production in the roots of L. japonicus by treatment with LPS of M. loti. LPS was prepared by phenol-hot water extraction and separated into several fractions: polysaccharide, lipooligosaccharide, oligosaccharide and lipid A. In the roots of L. japonicus, NO production was observed with an NO-specific fluorescent dye 4, 10 and 24 h after treatment with each fraction of LPS. NO production was detected 4 h after treatment with all fractions. NO production was also detectable 24 h after treatment, except after treatment with the polysaccharide and oligosaccharide fractions. Expression of a class 1 hemoglobin gene and application of an NO scavenger showed that the treatment with LPS and LOS induced a similar response to inoculation with M. loti. These data suggest that LPS of M. loti induces NO production after inoculation with M. loti.

Rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. The completion of several rhizobial genome sequences provides opportunities for genome-wide functional studies of the physiological roles of many rhizobial genes. In order to carry out genome-wide phenotypic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, by transposon mutagenesis. Transposon insertion mutants were generated using the signature-tagged mutagenesis (STM) technique and a total of 29,330 independent mutants were obtained. Along with the collection of transposon mutants, we have determined the transposon insertion sites for 7892 clones, and confirmed insertions in 3680 non-redundant M. loti genes (50.5% of the total number of M. loti genes). Transposon insertions were randomly distributed throughout the M. loti genome without any bias toward G+C contents of insertion target sites and transposon plasmids used for the mutagenesis. We also show the utility of STM mutants by examining the specificity of signature tags and test screenings for growth- and nodulation-deficient mutants. This defined mutant library allows for genome-wide forward- and reverse-genetic functional studies of M. loti and will serve as an invaluable resource for researchers to further our understanding of rhizobial biology.

Global viewing of protein-protein interactions (PPIs) is a useful way to assign biological roles to large numbers of proteins predicted by complete genome sequence. Here, we systematically analyzed PPIs in the nitrogen-fixing soil bacterium Mesorhizobium loti using a modified high-throughput yeast two-hybrid system. The aims of this study are primarily on the providing functional clues to M. loti proteins that are relevant to symbiotic nitrogen fixation and conserved in other rhizobium species, especially proteins with regulatory functions and unannotated proteins. By the screening of 1542 genes as bait, 3121 independent interactions involving 1804 proteins (24% of the total protein coding genes) were identified and each interaction was evaluated using an interaction generality (IG) measure and the general features of the interacting partners. Most PPIs detected in this study are novel interactions revealing potential functional relationships between genes for symbiotic nitrogen fixation and signal transduction. Furthermore, we have predicted the putative functions of unannotated proteins through their interactions with known proteins. The results described here represent new insight into protein network of M. loti and provide useful experimental clues to elucidate the biological function of rhizobial genes that can not be assigned directly from their genomic sequence.

Protein-protein interactions (PPIs) play crucial roles in protein function for a variety of biological processes. Data from large-scale PPI screening has contributed to understanding the function of a large number of predicted genes from fully sequenced genomes. Here, we report the systematic identification of protein interactions for the unicellular cyanobacterium Synechocystis sp. strain PCC6803. Using a modified high-throughput yeast two-hybrid assay, we screened 1825 genes selected primarily from (i) genes of two-component signal transducers of Synechocystis, (ii) Synechocystis genes whose homologues are conserved in the genome of Arabidopsis thaliana, and (iii) genes of unknown function on the Synechocystis chromosome. A total of 3236 independent two-hybrid interactions involving 1920 proteins (52% of the total protein coding genes) were identified and each interaction was evaluated using an interaction generality (IG) measure, as well as the general features of interacting partners. The interaction data obtained in this study should provide new insights and novel strategies for functional analyses of genes in Synechocystis, and, additionally, genes in other cyanobacteria and plant genes of cyanobacterial origin.

To better understand the molecular responses of plants to arbuscular mycorrhizal (AM) fungi, we analyzed the differential gene expression patterns of Lotus japonicus, a model legume, with the aid of a large-scale cDNA macroarray. Experiments were carried out considering the effects of contaminating microorganisms in the soil inoculants. When the colonization by AM fungi, i.e. Glomus mosseae and Gigaspora margarita, was well established, four cysteine protease genes were induced. In situ hybridization revealed that these cysteine protease genes were specifically expressed in arbuscule-containing inner cortical cells of AM roots. On the other hand, phenylpropanoid biosynthesis-related genes for phenylalanine ammonia-lyase (PAL), chalcone synthase, etc. were repressed in the later stage, although they were moderately up-regulated on the initial association with the AM fungus. Real-time RT-PCR experiments supported the array experiments. To further confirm the characteristic expression, a PAL promoter was fused with a reporter gene and introduced into L. japonicus, and then the transformants were grown with a commercial inoculum of G. mosseae. The reporter activity was augmented throughout the roots due to the presence of contaminating microorganisms in the inoculum. Interestingly, G. mosseae only colonized where the reporter activity was low. Comparison of the transcriptome profiles of AM roots and nitrogen-fixing root nodules formed with Mesorhizobium loti indicated that the PAL genes and other phenylpropanoid biosynthesis-related genes were similarly repressed in the two organs.

We characterized the expression profiles of LjHb1 and LjHb2, non-symbiotic hemoglobin (non-sym-Hb) genes of Lotus japonicus. Although LjHb1 and LjHb2 showed 77% homology in their cDNA sequences, LjHb2 is located in a unique position in the phylogenetic tree of plant Hbs. The 5'-upstream regions of both genes contain the motif AAAGGG at a position similar to that in promoters of other non-sym-Hb genes. Expression profiles obtained by using quantitative RT-PCR showed that LjHb1 and LjHb2 were expressed in all tissues of mature plants, and expression was enhanced in mature root nodules. LjHb1 was strongly induced under both hypoxic and cold conditions, and by the application of nitric oxide (NO) donor, whereas LjHb2 was induced only by the application of sucrose. LjHb1 was also induced transiently by the inoculation with the symbiotic rhizobium Mesorhizobium loti MAFF303099. Observations using fluorescence microscopy revealed the induction of LjHb1 expression corresponded to the generation of NO. These results suggest that non-sym-Hb and NO have important roles in stress adaptation and in the early stage of legume-rhizobium symbiosis.

Leguminous plants have both symbiotic and nonsymbiotic hemoglobin (sym- and nonsym-Hb) genes. Three symbiotic (LjLb1, 2, 3) and one nonsymbiotic (LjNSG1) Hb genes were isolated from a genomic library of Lotus japonicus MG20 Miyakojima. Two motif sequences (AAAGAT and CTCTT) critical for nodule specific expression were conserved on the 5'-upstream sequence of LjLb1, 2 and 3. The 5'-upstream region of LjNSG1 contained the sequence consensus for nonsym-Hb. RT-PCR with specific primer sets for each LjLb gene showed that all the sym-Hb genes (LjLb1, 2, 3) were expressed specifically and strongly in root nodules. The expression of LjLb1, 2 and 3 could not be detected in root, leaf or stem of a mature plant, whereas low level expression was detected in seedlings by RT-PCR. This suggests that sym-Hbs may have another unknown function besides being oxygen transporter for the microsymbiont in symbiotic nitrogen fixation. The expression of LjNSG1, examined with RT-PCR, was detected at low level in root, leaf and stem. The expression of LjNSG1 was enhanced in root nodules, whereas it was repressed in roots colonized by mycorrhizal fungi Glomus sp. R10. The repression of the nonsym-Hb gene was also observed in the roots of Medicago sativa colonized by Glomus sp. R10.

To evidence the notion that gangliosides involve neuronal cell interactions in the brain, we surveyed the presence of ganglioside-binding proteins in membrane lysates of adult rat cerebellum. Three proteins (p58, p90, and p160) were identified as GT1b-binding proteins by incubation of the blot of the membrane lysate with GT1b micelles. We generated a monoclonal antibody (mAb) specific to the polypeptide portion of the GT1b-binding proteins (YAK-2). The YAK-2 mAb specifically reacted with all three proteins on blots of proteins pretreated under nonreducing conditions for SDS-PAGE, but reacted mainly with p58 under reducing conditions, showing that p90 and p160 are oligomeric forms of p58. The binding activity of the YAK-2 mAb was completely inhibited by the presence of GT1b micelles, indicating the specificity of YAK-2 mAb for p58 and its oligomers. Immunohistochemical investigations revealed that both p58 and GT1b colocalize within the granular layer of adult rat cerebellum. Expression cloning of p58 cDNA was performed using YAK-2 mAb, and five putative clones were obtained. Among them, the nucleotide sequence of one cDNA completely matched that of rat brain-specific sodium-dependent inorganic phosphate cotransporter (rBNPI), a 61 kDa membrane protein. COS7 cells were transfected with a Flag-chimeric construct containing the rBNPI/p58 cDNA, and the membrane lysate was subjected to immunoprecipitation with anti-Flag antibody. One protein (64 kDa) was detected only with YAK-2 mAb, and the membrane lysate specifically bound to GT1b micelles. Taking together, we propose that rBNPI/p58 functions as a GT1b-binding protein in neuronal cells.