Intracranial dural arteriovenous fistula mimicking neuromyelitis optica spectrum disorder: A case report.

Neuromyelitis optica spectrum disorder (NMOSD) is an immune-mediated disorder. It often develops acute myelopathy due to longitudinally extensive transverse myelitis (LETM), although other disorders can cause an LETM-like lesion. Here, we report a 76-year-old patient presenting with acute-onset, progressive myelopathy, which proved to be caused by an intracranial dural arteriovenus fistula (dAVF). Magnetic resonance imaging (MRI) revealed a longitudinally extensive spinal cord lesion, which was further extended rostrally to the medulla. Although cord surface flow voids were absent on T2-weighted MRI, abnormally congested peri-spinal veins showed up with gadolinium contrast. Angiography confirmed dAVF in the posterior fossa, which drained into the peri-spinal veins. Intracranial dAVF should be considered as a differential diagnosis of NMOSD, because it is not immunologically but is surgically treatable.

Dupilumab for Atopic Dermatitis, a Possible Risk Factor of Juvenile Ischemic Stroke: A Case Report.

Dupilumab, a dual inhibitor of IL-4 and IL-13 cytokine signaling, is indicated for the treatment of moderate-to-severe atopic dermatitis, which leads to the control of atopic dermatitis. The cytokines IL-4 and IL-13 are related to vascular inflammation, which is mediated by vascular endothelial cells. We report the case of a 20-year-old man with atopic dermatitis treated with dupilumab for half a year, who presented with sudden onset of dizziness, nausea, and slight cerebellar ataxia. Brain magnetic resonance imaging revealed acute infarction in the bicerebellar hemispheres. No risk factors known to be associated with ischemic stroke in young adults were detected. We suspected this ischemic stroke might be related to dupilumab. The administration of dupilumab was discontinued, and he had no recurrence subsequently. IL-4 and IL-13, anti-inflammatory cytokines secreted from T helper 2 cells, suppress proinflammatory cytokines. Therefore, dupilumab, a dual inhibitor of IL-4 and IL-13 cytokine signaling, leads to the promotion of coagulation and thrombosis. We speculate that the activation of proinflammatory cytokines in vascular endothelial cells by the inhibition of IL-4 and IL-13 signaling by dupilumab led to ischemic stroke even at a young age.

Training of Intra-Axial Brain Tumor Resection Using a Self-Made Simple Device with Agar and Gelatin.

INTRODUCTION: Self-made devices composed of agar and gelatin gel were used for resident training in intra-axial brain tumor resection. The mixture gel of agar and gelatin is retractable and can be suctioned. Hardness of the gel depends on the concentration of the solution. Therefore, by changing the concentration, it is easy to make gels of various hardness. METHODS: In this study, a mass of gel that looked like a tumor was placed into another gel that looked like the brain. A part of the "brain" was regarded as the eloquent area. Three types of "tumor" were prepared: hard, moderately hard, and soft tumors. Residents tried to remove the tumor entirely with minimal brain invasion. The training was repeated with 3 types of gel. After resection, the weight of the residual tumor, resected normal brain, and resected eloquent area were measured, and the time taken for removal was recorded. RESULTS: These data were compared between residents and neurosurgeons. We also analyzed how these data improved with repeated practice. In most cases, residual tumor, resected normal brain, resected eloquent area, and time taken for removal were less in neurosurgeons than in residents. Repeated training made residents more skillful. The responses of the trainees were almost all favorable. CONCLUSIONS: Our devices with "tumors" of various hardness appear to be suitable for resident training in each surgical skill. For the next step of this study, we will attempt to fabricate more practical 3-dimensional gel models for presurgical simulation.

Quantitative targeted absolute proteomics for 28 human transporters in plasma membrane of Caco-2 cell monolayer cultured for 2, 3, and 4 weeks.

The purpose of the present study was to evaluate and compare the absolute protein expression levels of 28 drug-related transporters in Caco-2 cell monolayers cultured for 2, 3, and 4 weeks. Plasma membrane fractions of Caco-2 cells cultured on Transwell inserts for 2, 3 and 4 weeks were prepared and digested with trypsin, and then simultaneous absolute quantification of 28 transporters and Na(+)/K(+)-ATPase was conducted using our established quantitative targeted absolute proteomic technique. Nine transporters and Na(+)/K(+)-ATPase were detected. MDR1, BCRP, PEPT1, OSTα and OSTß were highly expressed (greater than 1 fmol/µg protein), while MRP2, MRP4, OATP2B1 and MCT1 were moderately expressed (0.328-0.871 fmol/µg protein). No significant difference was observed in the protein expression levels of these transporters or Na(+)/K(+)-ATPase among the 2-, 3- and 4-week cultures. The other 19 transporters, including MRP1, MRP3, OATP1A2, OATP3A1, OATP4A1, and OATP1B3, were not detected. This information about the rank order of transporter protein expression will be useful to predict what transporter(s) are likely or unlikely to influence the permeability of various compounds across monolayers of Caco-2 cells, which are widely used in drug development studies.

The roles of the dimeric and tetrameric structures of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria.

The molecular machinery of the cyanobacterial circadian clock consists of three proteins, KaiA, KaiB, and KaiC. The three Kai proteins interact with each other and generate circadian oscillations in vitro in the presence of ATP (an in vitro KaiABC clock system). KaiB consists of four subunits organized as a dimer of dimers, and its overall shape is that of an elongated hexagonal plate with a positively charged cleft flanked by two negatively charged ridges. We found that a mutant KaiB with a C-terminal deletion (KaiB(1-94)), which lacks the negatively charged ridges, was a dimer. Despite its dimeric structure, KaiB(1-94) interacted with KaiC and generated normal circadian oscillations in the in vitro KaiABC clock system. KaiB(1-94) also generated circadian oscillations in cyanobacterial cells, but they were weak, indicating that the C-terminal region and tetrameric structure of KaiB are necessary for the generation of normal gene expression rhythms in vivo. KaiB(1-94) showed the highest affinity for KaiC among the KaiC-binding proteins we examined and inhibited KaiC from forming a complex with SasA, which is involved in the main output pathway from the KaiABC clock oscillator in transcription regulation. This defect explains the mechanism underlying the lack of normal gene expression rhythms in cells expressing KaiB(1-94).

Functionally important structural elements of the cyanobacterial clock-related protein Pex.

Pex, a clock-related protein involved in the input pathway of the cyanobacterial circadian clock system, suppresses the expression of clock gene kaiA and lengthens the circadian period. Here, we determined the crystal structure of Anabaena Pex (AnaPex; Anabaena sp. strain PCC 7120) and Synechococcus Pex (SynPex; Synechococcus sp. strain PCC 7942). Pex is a homodimer that forms a winged-helix structure. Using the DNase I protection and electrophoresis mobility shift assays on a Synechococcus kaiA upstream region, we identified a minimal 25-bp sequence that contained an imperfectly inverted repeat sequence as the Pex-binding sequence. Based on crystal structure, we predicted the amino acid residues essential for Pex's DNA-binding activity and examined the effects of various Ala-substitutions in the alpha3 helix and wing region of Pex on in vitro DNA-binding activity and in vivo rhythm functions. Mutant AnaPex proteins carrying a substitution in the wing region displayed no specific DNA-binding activity, whereas those carrying a substitution in the alpha3 helix did display specific binding activity. But the latter were less thermostable than wild-type AnaPex and their in vitro functions were defective. We concluded that Pex binds a kaiA upstream DNA sequence via its wing region and that its alpha3 helix is probably important to its stability.

ATPase activity and its temperature compensation of the cyanobacterial clock protein KaiC.

KaiA, KaiB and KaiC constitute the circadian clock machinery in cyanobacteria. KaiC is a homohexamer; its subunit contains duplicated halves, each with a set of ATPase motifs. Here, using highly purified KaiC preparations of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 produced in Escherichia coli, we found that the N- and C-terminal domains of KaiC had extremely weak ATPase activity. ATPase activity showed temperature compensation in wild-type KaiC, but not in KaiC(S431A/T432A), a mutant that lacks two phosphorylation sites. We concluded that KaiC phosphorylation is involved in the ATPase temperature-compensation mechanism-which is probably critical to the stability of the circadian clock in cyanobacteria-and we hypothesized the following temperature-compensation mechanism: (i) The C-terminal phosphorylation sites of a KaiC hexamer subunit are phosphorylated by the C-terminal domain of an adjacent KaiC subunit; (ii) the phosphorylation suppresses the ATPase activity of the C-terminal domain; and (iii) the phosphorylated KaiC spontaneously dephosphorylates, resulting in the recover of ATPase activity.

Quantitative atlas of membrane transporter proteins: development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria.

PURPOSE: To develop an absolute quantification method for membrane proteins, and to construct a quantitative atlas of membrane transporter proteins in the blood-brain barrier, liver and kidney of mouse. METHODS: Mouse tissues were digested with trypsin, and mixed with stable isotope labeled-peptide as a quantitative standard. The amounts of transporter proteins were simultaneously determined by liquid chromatography-tandem mass spectrometer (LC/MS/MS). RESULTS: The target proteins were digested in-silico, and target peptides for analysis were chosen on the basis of the selection criteria. All of the peptides selected exhibited a detection limit of 10 fmol and linearity over at least two orders of magnitude in the calibration curve for LC/MS/MS analysis. The method was applied to obtain the expression levels of 34 transporters in liver, kidney and blood-brain barrier of mouse. The quantitative values of transporter proteins showed an excellent correlation with the values obtained with existing methods using antibodies or binding molecules. CONCLUSION: A sensitive and simultaneous quantification method was developed for membrane proteins. By using this method, we constructed a quantitative atlas of membrane transporter proteins at the blood-brain barrier, liver and kidney in mouse. This technology is expected to have major implications for various fields of biomedical science.

Hexamerization by the N-terminal domain and intersubunit phosphorylation by the C-terminal domain of cyanobacterial circadian clock protein KaiC.

Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. The opposing spherical regions of the dumbbell-shaped structures correspond to the N-terminal and C-terminal domains of KaiC. Previously, we hypothesized that the N-terminal domain of KaiC is responsible for the ATP-induced hexamerization of KaiC while the C-terminal domain is responsible for the phosphorylation of KaiC (Hayashi et al. 2004, J. Biol. Chem. 279, 52331-52337). Here, we tested that hypothesis using the purified protein of each domain. We prepared N-terminal and C-terminal domain proteins (KaiCN and KaiCC, respectively), examined their function by analyzing their ATP- or 5'-adenylylimidodiphosphate (AMPPNP; an unhydrolyzable ATP analog)-induced hexamerization, interactions with KaiA, and phosphorylation, and we demonstrated the following: (1) KaiCN had higher ATP- or AMPPNP-induced oligomerization activity than KaiCC. (2) KaiCc had phosphorylation activity as KaiCWT whereas KaiCN had no activity. (3) KaiCC interacted with KaiA whereas KaiCN did not. (4) The interactions of KaiCC with KaiA did not require that KaiC has a hexamer structure. (5) The interactions of KaiCC with KaiA enhanced the phosphorylation of KaiCC. Furthermore, we presented evidence for the intersubunit phosphorylation of KaiC. KaiCCatE2-, which lacks KaiC phosphorylation activity due to mutations of the catalytic Glu residues, was phosphorylated when it was co-incubated with KaiCC. We propose that the KaiC hexamer consists of a rigid ring structure formed by six N-terminal domains with hexamerization activity and a flexible structure formed by six C-terminal domains with intersubunit phosphorylation activity.

Functionally important substructures of circadian clock protein KaiB in a unique tetramer complex.

KaiB is a component of the circadian clock molecular machinery in cyanobacteria, which are the simplest organisms that exhibit circadian rhythms. Here we report the x-ray crystal structure of KaiB from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The KaiB crystal diffracts at a resolution of 2.6 A and includes four subunits organized as a dimer of dimers, each composed of two non-equivalent subunits. The overall shape of the tetramer is an elongated hexagonal plate, with a single positively charged cleft flanked by two negatively charged ridges whose surfaces includes several terminal chains. Site-directed mutagenesis of Synechococcus KaiB confirmed that alanine substitution of residues Lys-11 or Lys-43 in the cleft, or deletion of C-terminal residues 95-108, which forms part of the ridges, strongly weakens in vivo circadian rhythms. Characteristics of KaiB deduced from the x-ray crystal structure were also confirmed by physicochemical measurements of KaiB in solution. These data suggest that the positively charged cleft and flanking negatively charged ridges in KaiB are essential for the biological function of KaiB in the circadian molecular machinery in cyanobacteria.

Roles of two ATPase-motif-containing domains in cyanobacterial circadian clock protein KaiC.

Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. Each subunit has duplicated domains, and each domain has a set of ATPase motifs. The two spherical regions of the dumbbell are likely to correspond to two domains. We examined the role of the two sets of ATPase motifs by analyzing the in vitro activity of ATPgammaS binding, AMPPNP-induced hexamerization, thermostability, and phosphorylation of KaiC and by in vivo rhythm assays both in wild type KaiC (KaiCWT) and KaiCs carrying mutations in either Walker motif A or deduced catalytic Glu residues. We demonstrated that 1) the KaiC subunit had two types of ATP-binding sites, a high affinity site in N-terminal ATPase motifs and a low affinity site in C-terminal ATPase motifs, 2) the N-terminal motifs were responsible for hexamerization, and 3) the C-terminal motifs were responsible for both stabilization and phosphorylation of the KaiC hexamer. We proposed the following reaction mechanism. ATP preferentially binds to the N-terminal high affinity site, inducing the hexamerization of KaiC. Additional ATP then binds to the C-terminal low affinity site, stabilizing and phosphorylating the hexamer. We discussed the effect of these KaiC mutations on circadian bioluminescence rhythm in cells of cyanobacteria.

Crystallization and preliminary crystallographic analysis of the circadian clock protein KaiB from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1.

KaiB is a component of the circadian clock oscillator in cyanobacteria, which are the simplest organisms that exhibit circadian rhythms. KaiB consists of 108 amino-acid residues and has a molecular weight of 12 025 Da. KaiB and Cys-substituted KaiB mutants from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 were expressed as GST-fusion proteins in Escherichia coli, purified and crystallized. The crystals of wild-type KaiB belong to the monoclinic space group P2(1), with unit-cell parameters a = 89.6, b = 71.2, c = 106.8 A, beta = 100.1 degrees. While the native crystals diffract to 3.7 A, osmium derivatives, which show an approximately 4 A shrinkage in the b axis, diffract to 2.6 A. The crystals of the singly Cys-substituted mutant T64C with Hg, which show different morphology, diffract to 2.5 A and belong to the monoclinic space group P2, with unit-cell parameters a = 63.7, b = 33.4, c = 93.7 A, beta = 100.1 degrees. Anomalous difference Patterson maps of the Os- and Hg-derivative crystals had significant peaks in their Harker sections, suggesting that both derivatives are suitable for structure determination.

Stoichiometric interactions between cyanobacterial clock proteins KaiA and KaiC.

We determined the stoichiometry of KaiA-KaiC interactions. Using immunoblotting and two-dimensional Native- and SDS-PAGE (2DNS-PAGE) analysis, we demonstrated that the reaction products of KaiA-KaiC interactions in the presence of ATP consisted of only phosphorylated KaiC whereas in the presence of the unhydrolyzable analogue 5'-adenylylimidodiphosphate (AMPPNP) they consisted of KaiA and KaiC. In the presence of ATP, the KE (molar ratio of KaiA dimer to KaiC hexamer giving half saturation in the enhancement of KaiC phosphorylation) was 0.25, and IAsys affinity biosensor analysis demonstrated that 1 molecule of KaiA dimer interacted with 1 molecule of KaiC hexamer. In the presence of AMPPNP, the ratio of KaiA dimer to KaiC hexamer in KaiA-KaiC complexes was determined to be 2 by 2DNS-PAGE, Native-PAGE/Scatchard plot, and IAsys analyses. These results suggest that 2 molecules of KaiA dimer can interact with 1 molecule of KaiC hexamer, and that interactions of at least 1 molecule of KaiA dimer with 1 molecule of KaiC hexamer are enough to enhance the phosphorylation of KaiC by KaiA at an almost saturated level.

ATP-induced hexameric ring structure of the cyanobacterial circadian clock protein KaiC.

BACKGROUND: KaiA, KaiB and KaiC are cyanobacterial circadian clock proteins. KaiC contains two ATP/GTP-binding Walker's motif As, and mutations in these regions affect the clock oscillations. RESULTS: ATP induced the hexamerization of KaiC. The Km value for the ATP for the hexamerization was 1.9 micro m. Triphosphate nucleotides bound to the two Walker's motif As, and their binding functioned cooperatively for the hexamerization. An unhydrolysable substrate, 5'-adenylylimidodiphosphate (AMPPNP), also induced the hexamerization, indicating that nucleotide binding, but not its hydrolysis, is essential for the hexamerization. Mutations in each of the two Walker's motif As that affect the clock phenotype increased the Km value for ATP and inhibited the hexamerization. Thus, the KaiC hexamerization seems to be necessary for its clock function. The KaiC hexamer has the shape of a hexagonal pot with a diameter and height of approximately 100 A and with a relatively large cavity (73 A deep and 18-34 A wide) inside. This pot-shaped structure suggests that KaiC functions in a similar manner to F1-ATPase, helicase or ATP-dependent protease/chaperon, all of which have dynamic activities inside the central cavity of their hexameric rings. CONCLUSION: ATP-induced KaiC hexamerization is necessary for the clock function of KaiC.