In JoVE (2)

Other Publications (55)

Articles by Shigeki Watanabe in JoVE

Other articles by Shigeki Watanabe on PubMed

[Pulmonary Infection Caused by Mycobacterium Szulgai: a Case Report]

Kekkaku : [Tuberculosis]. May, 2002  |  Pubmed ID: 12073621

We reported a case of pulmonary infection caused by Mycobacterium szulgai (M. szulgai) in an immunocompetent, asymptomatic 55-year-old man without underlying disease. A chest radiograph of an annual health examination revealed a right upper lobe infiltrate with thin-walled cavities, which was not present in the previous year. An acid-fast stain of bronchial washing fluid was positive, and antimycobacterial chemotherapy with isoniazid (400 mg/day), rifampin (450 mg/day), and ethambutol (750 mg/day) was initiated on presumptive diagnosis of the case as tuberculosis. DNA-DNA hybridization of sputum and bronchial washing samples identified M. szulgai as the causative organism. Antimicrobial susceptibility testing indicated that the isolate was sensitive to most common antimycobacterial drugs except capreomycin (CPM) and p-aminosalicylic acid (PAS), and was also sensitive to clarithromycin and fluoroquinolones including ofloxacin, levofloxacin, sparfloxacin, and ciprofloxacin. After 12 months of therapy, a repeat chest radiograph demonstrated improvement of the right upper lobe infiltrate. When M. szulgai is isolated, it almost always represents a true pathogen. Therefore, the detection of even a small number of M. szulgai warrants treatment based on susceptibility testing.

[Role of Cholecystokinin Receptor in Methamphetamine-induced Reverse Tolerance]

Nihon Shinkei Seishin Yakurigaku Zasshi = Japanese Journal of Psychopharmacology. Oct, 2002  |  Pubmed ID: 12451697

[Role of Brain Prostaglandin Biosynthesis in Behavioral Disorders Induced by Tetrahydrocannabinol in Marihuana]

Nihon Shinkei Seishin Yakurigaku Zasshi = Japanese Journal of Psychopharmacology. Oct, 2002  |  Pubmed ID: 12451699

[A Case Report of Esophageal Small Cell Carcinoma: a Long-term Survival by Radio-chemotherapy with Carboplatin and Etoposide]

Nihon Shokakibyo Gakkai Zasshi = The Japanese Journal of Gastro-enterology. Nov, 2004  |  Pubmed ID: 15570870

[Refractory Evans Syndrome Complicated with Transverse Sinus Thrombosis]

[Rinshō Ketsueki] The Japanese Journal of Clinical Hematology. Jul, 2005  |  Pubmed ID: 16440744

A 29-year-old woman was diagnosed as having Evans syndrome in 2002 and underwent a splenectomy for the refractory status of the disorder in May 2004. One and a half months after the operation, her platelet count again decreased due to relapse, and she was then prescribed with high dose dexamethasone (38 mg/day x 4 days). Five days after the medication, she complained of a severe headache and then fell into coma, even though her platelet count had risen to 8 x 10(4)/mm3. Computer tomography scan of the brain showed severe edema with a massive hemorrhage in left temporooccipital lobe, which was compatible with cerebral transverse sinus thrombosis. After resection of the damaged brain, her level of consciousness gradually recovered, although visual disturbance and moderate hemiplegia remained. This is the fourth case of idiopathic thrombocytopenic purpura which was complicated with sinus thrombosis in the literature.

[Epstein-Barr Virus Related B-cell Lymphoproliferative Disorder Complicated with Bone Marrow Fibrosis, Which Was Successfully Treated with 2 Courses of CHOP Regimen]

[Rinshō Ketsueki] The Japanese Journal of Clinical Hematology. Oct, 2005  |  Pubmed ID: 16440776

A 68-year-old man was referred to our hospital in August 2003 with a high fever, a prominent inflammatory reaction in blood test and also bicytopenia (anemia and thrombocytopenia) with marked hepatosplenomegaly. He was temporarily diagnosed as having malignant lymphoma considering the elevated levels of LDH and soluble interleukin-2 receptor, following which treatment with the CHOP regimen was started. Thereafter, based on the pathological findings from a bone marrow biopsy and a quite high viral load revealed by real-time PCR analysis, the diagnosis was changed to Epstein-Barr virus (EBV) related B-lymphoproliferative disorder (B-LPD) complicated with reticulin fibrosis. A total of 2 courses of the CHOP regimen together with anti-viral reagents almost resolved the clinical symptoms and abnormal findings of laboratory tests. This unique case was considered to be "a senile EBV positive B-LPD" complicated with a secondary myelofibrosis, a category of the disorder which has recently been proposed by Shigeo Nakamura at the Aichi Cancer Center.

[A Case of Severe Adult-onset Still' S Disease Presenting with Pleuropericarditis]

Nihon KokyÅ«ki Gakkai Zasshi = the Journal of the Japanese Respiratory Society. May, 2006  |  Pubmed ID: 16780097

Adult-onset Still' s disease (AOSD) is an uncommon rheumatic disease characterized by high spiking fever, arthritis, an evanescent skin rash and variety of systemic symptoms, though initial onset of pleuropulmonary manifestation is relatively rare and could be responsible for a delay in diagnosis. We report a case of AOSD presenting with pleuritis with concomitant pericardial effusion. A 42-year-old Japanese man was admitted with a spiking fever of 40 degrees C, hyperleucocytosis (21.6 x 10(9)/l), and a high titer of C-reactive protein (16.84mg/dl). Chest X-ray film and computed tomography showed bilateral pleural effusion and massive pericardial effusion which both required tube drainage. Analyses of fluids revealed that both were exudative and sterile, and pleural biopsy showed nonspecific inflammation with mild fibrosis. Neither antibiotics nor antituberculosis drugs were effective. Rash, hepatosplenomegaly, polyarthritis, pharyngitis and right hypochondralgia were accompanied by serum hyperferritinemia. After exclusion of the possibility of infection, other connective tissue disease and malignancy, a diagnosis of AOSD was made. Improvement was not observed with nonsteroidal anti-inflammatory drug and corticosteroid therapy. Double filtration plasmapheresis (DFPP) following steroid pulse therapy alleviated the symptoms and the laboratory data immediately and corticosteroids could be tapered. DFPP is a safe therapeutic procedure and can be an alternative for refractory AOSD.

Health-related Quality of Life Among Japanese Women with Iron-deficiency Anemia

Quality of Life Research : an International Journal of Quality of Life Aspects of Treatment, Care and Rehabilitation. Dec, 2006  |  Pubmed ID: 16826432

Iron-deficiency anemia (IDA) is a common disease in females of childbearing age. Although iron supplementation quickly improves laboratory-measured parameters, its effect on health-related quality of life is unknown. Here, we conducted a prospective follow-up study to evaluate health-related quality of life in pre-menopausal women diagnosed with IDA. A convenience sample of 92 patients who visited Tokai University Hospital and three other affiliated hospitals were asked to fill out the Medical Outcome Study 36-item short-form health survey (SF-36) during the course of treatment (baseline, and 1 and 3 months after the start of treatment). At baseline, vitality and general health scores were significantly lower than the Japanese national norms. After the start of therapy, however, a significant improvement was seen in all domain scores except role emotional (RE), and at 3 months all eight scores were comparable to or greater than the national norms. In particular, physical functioning and vitality scores of patients with a lower hemoglobin level ( < 9.0 g/dl) at baseline showed a dramatic improvement. Iron supplementation in IDA patients improves not only hemoglobin levels, but also physical function, vitality, and general health perception.

UNC-46 is Required for Trafficking of the Vesicular GABA Transporter

Nature Neuroscience. Jul, 2007  |  Pubmed ID: 17558401

Mutations in unc-46 in Caenorhabditis elegans cause defects in all behaviors that are mediated by GABA. Here we show that UNC-46 is a sorting factor that localizes the vesicular GABA transporter to synaptic vesicles. The UNC-46 protein is related to the LAMP (lysosomal associated membrane protein) family of proteins and is localized at synapses. In unc-46 mutants, the vesicular transporter is not found specifically in synaptic vesicles but rather is diffusely spread along the axon. Mislocalization of the transporter severely reduces the frequency of miniature currents, but the remaining currents are normal in amplitude. Because the number of synaptic vesicles is not depleted, it is likely that only a fraction of vesicles harbor the transporter in unc-46 mutants. Our data indicate that the transporter and UNC-46 have mutual roles in sorting. The vesicular GABA transporter recruits UNC-46 to synaptic vesicle precursors in the cell body, and UNC-46 sorts the transporter at the cell body and during endocytosis at the synapse.

Open Syntaxin Docks Synaptic Vesicles

PLoS Biology. Aug, 2007  |  Pubmed ID: 17645391

Synaptic vesicles dock to the plasma membrane at synapses to facilitate rapid exocytosis. Docking was originally proposed to require the soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins; however, perturbation studies suggested that docking was independent of the SNARE proteins. We now find that the SNARE protein syntaxin is required for docking of all vesicles at synapses in the nematode Caenorhabditis elegans. The active zone protein UNC-13, which interacts with syntaxin, is also required for docking in the active zone. The docking defects in unc-13 mutants can be fully rescued by overexpressing a constitutively open form of syntaxin, but not by wild-type syntaxin. These experiments support a model for docking in which UNC-13 converts syntaxin from the closed to the open state, and open syntaxin acts directly in docking vesicles to the plasma membrane. These data provide a molecular basis for synaptic vesicle docking.

[Supplementary Motor Area Syndrome with Frontal Glioma]

Brain and Nerve = Shinkei KenkyÅ« No Shinpo. Jul, 2007  |  Pubmed ID: 17663151

The supplementary motor area (SMA) is a region located within each cerebral hemisphere at the posterior medial border of the frontal lobe. It is considered to play an important role in planning, initiating and maintaining sequential motor actions. In this report, we aimed to confirm or invalidate the somatotopic organization of the SMA, correlates the pattern of clinical symptoms observed after SMA removal with the extent of resection. Althogh there was no apparent change shown in the monitoring of intraoperative motor evoked potential (MEP), four patients displayed postoperative SMA syndrome on the side of the body contralateral to the SMA resection. All patients developed postoperative severe hemiplegia. One dominant frontal glioma patient was followed by transient mutism and motor aphasia. In this study, there is no correlation between extent of SMA resection and postoperative clinical pattern of deficits.

[Two Cases of Herpes Simplex Esophagitis During Treatment for Lung Cancer]

Nihon KokyÅ«ki Gakkai Zasshi = the Journal of the Japanese Respiratory Society. Jul, 2007  |  Pubmed ID: 17682465

Herpes simplex virus (HSV) is one of the three major causes of infectious esophagitis, along with Candida albicans and Cytomegalo virus (CMV). Most cases occur in immunocompromised hosts, in whom this can be life threatening. We report two cases of herpes simplex esophagitis occurring during treatment for lung cancer. Case 1: An 80-year-old man with radiation pneumonia caused by radiotherapy for lung cancer was admitted for treatment with antibiotics and corticosteroids. Shortly after initiation of treatment, he complained of dysphasia. Endoscopic examination revealed herpes simplex esophagitis. Case 2: A 71-year-old man was given corticosteroids for cryptogenic organizing pneumonia following chemotherapy for lung cancer. During treatment, the patient complained of odynophagia. Endoscopic examination revealed herpes simplex esophagitis. Both cases died due to progression of lung cancer and acute respiratory distress syndrome, despite administration of acyclovir. When immunocompromised patients complain of prolonged dysphagia and odynophagia, the presence of herpes simplex esophagitis should be clarified by endoscopic examination. It is occasionally difficult to distinguish between HSV and Candida esophagitis by endoscopic observation alone. Esophageal mucosal endoscopic cytology can help differentiate between these three infectious agents.

UNC-80 and the NCA Ion Channels Contribute to Endocytosis Defects in Synaptojanin Mutants

Current Biology : CB. Sep, 2007  |  Pubmed ID: 17825559

Synaptojanin is a lipid phosphatase required to degrade phosphatidylinositol 4,5 bisphosphate (PIP(2)) at cell membranes during synaptic vesicle recycling. Synaptojanin mutants in C. elegans are severely uncoordinated and are depleted of synaptic vesicles, possibly because of accumulation of PIP(2). To identify proteins that act downstream of PIP(2) during endocytosis, we screened for suppressors of synaptojanin mutants in the nematode C. elegans. A class of uncoordinated mutants called "fainters" partially suppress the locomotory, vesicle depletion, and electrophysiological defects in synaptojanin mutants. These suppressor loci include the genes for the NCA ion channels, which are homologs of the vertebrate cation leak channel NALCN, and a novel gene called unc-80. We demonstrate that unc-80 encodes a novel, but highly conserved, neuronal protein required for the proper localization of the NCA-1 and NCA-2 ion channel subunits. These data suggest that activation of the NCA ion channel in synaptojanin mutants leads to defects in recycling of synaptic vesicles.

CAPS and Syntaxin Dock Dense Core Vesicles to the Plasma Membrane in Neurons

The Journal of Cell Biology. Feb, 2008  |  Pubmed ID: 18250196

Docking to the plasma membrane prepares vesicles for rapid release. Here, we describe a mechanism for dense core vesicle docking in neurons. In Caenorhabditis elegans motor neurons, dense core vesicles dock at the plasma membrane but are excluded from active zones at synapses. We have found that the calcium-activated protein for secretion (CAPS) protein is required for dense core vesicle docking but not synaptic vesicle docking. In contrast, we see that UNC-13, a docking factor for synaptic vesicles, is not essential for dense core vesicle docking. Both the CAPS and UNC-13 docking pathways converge on syntaxin, a component of the SNARE (soluble N-ethyl-maleimide-sensitive fusion protein attachment receptor) complex. Overexpression of open syntaxin can bypass the requirement for CAPS in dense core vesicle docking. Thus, CAPS likely promotes the open state of syntaxin, which then docks dense core vesicles. CAPS function in dense core vesicle docking parallels UNC-13 in synaptic vesicle docking, which suggests that these related proteins act similarly to promote docking of independent vesicle populations.

Mu2 Adaptin Facilitates but is Not Essential for Synaptic Vesicle Recycling in Caenorhabditis Elegans

The Journal of Cell Biology. Dec, 2008  |  Pubmed ID: 19047463

Synaptic vesicles must be recycled to sustain neurotransmission, in large part via clathrin-mediated endocytosis. Clathrin is recruited to endocytic sites on the plasma membrane by the AP2 adaptor complex. The medium subunit (micro2) of AP2 binds to cargo proteins and phosphatidylinositol-4 ,5-bisphosphate on the cell surface. Here, we characterize the apm-2 gene (also called dpy-23), which encodes the only micro2 subunit in the nematode Caenorhabditis elegans. APM-2 is highly expressed in the nervous system and is localized to synapses; yet specific loss of APM-2 in neurons does not affect locomotion. In apm-2 mutants, clathrin is mislocalized at synapses, and synaptic vesicle numbers and evoked responses are reduced to 60 and 65%, respectively. Collectively, these data suggest AP2 micro2 facilitates but is not essential for synaptic vesicle recycling.

Differential Requirements for Clathrin in Receptor-mediated Endocytosis and Maintenance of Synaptic Vesicle Pools

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2009  |  Pubmed ID: 19151157

Clathrin is a coat protein involved in vesicle budding from several membrane-bound compartments within the cell. Here we present an analysis of a temperature-sensitive (ts) mutant of clathrin heavy chain (CHC) in a multicellular animal. As expected Caenorhabditis elegans chc-1(b1025ts) mutant animals are defective in receptor-mediated endocytosis and arrest development soon after being shifted to the restrictive temperature. Steady-state clathrin levels in these mutants are reduced by more than 95% at all temperatures. Hub interactions and membrane associations are lost at the restrictive temperature. chc-1(b1025ts) animals become paralyzed within minutes of exposure to the restrictive temperature because of a defect in the nervous system. Surprisingly synaptic vesicle number is not reduced in chc-1(b1025ts) animals. Consistent with the normal number of vesicles, postsynaptic miniature currents occur at normal frequencies. Taken together, these results indicate that a high level of CHC activity is required for receptor-mediated endocytosis in nonneuronal cells but is largely dispensable for maintenance of synaptic vesicle pools.

[A Case of Cryptococcal Pneumonia Accompanying Meningitis]

Nihon KokyÅ«ki Gakkai Zasshi = the Journal of the Japanese Respiratory Society. Jan, 2009  |  Pubmed ID: 19198229

Cryptococcosis is a fungal infection caused by cryptococcus neoformans. Cryptococcal pneumonia occurs due to inhalation of the organism into the respiratory tract, sometimes accompanied by meningitis in immunocompromised patients, and can be life-threatening. We report a case of cryptococcal meningitis occurring during corticosteroid therapy for rheumatoid arthritis. Case: A 82-year-old woman with rheumatoid arthritis was given a diagnosis of cryptococcal meningitis, and improved after administeration of amphotericin B in combination with flucytosine. However 3 weeks later, side effects occurred, she was given fluconazole alone, but her condition worsened and she died. In severe cases of cryptococcal meningitis, we should take into account drug susceptibility tests and drug concentrations at the site of infection.

Evaluation of (64)Cu-labeled DOTA-D-Phe(1)-Tyr (3)-octreotide ((64)Cu-DOTA-TOC) for Imaging Somatostatin Receptor-expressing Tumors

Annals of Nuclear Medicine. Aug, 2009  |  Pubmed ID: 19504168

In-111 ((111)In)-labeled octreotide has been clinically used for imaging somatostatin receptor-positive tumors, and radiolabeled octreotide analogs for positron emission tomography (PET) have been developed. Cu-64 ((64)Cu; half-life, 12.7 h) is an attractive radionuclide for PET imaging and is produced with high specific activity using a small biomedical cyclotron. The aim of this study is to produce and fundamentally examine a (64)Cu-labeled octreotide analog, (64)Cu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-D: -Phe(1)-Tyr(3)-octreotide ((64)Cu-DOTA-TOC).

Chelating Ion-exchange Methods for the Preparation of No-carrier-added 64Cu

Nuclear Medicine and Biology. Aug, 2009  |  Pubmed ID: 19647164

We have developed a method for producing no-carrier-added (64)Cu by using chelating resin bearing iminodiacetic acid groups.

A Case of Acquired Aplastic Anemia with Repeated Cerebral Infarctions at the Beginning of Immunosuppressive Therapy

The Tokai Journal of Experimental and Clinical Medicine. Sep, 2009  |  Pubmed ID: 21319000

Acquired aplastic anemia is a rare hematopoietic stem-cell disorder that results in pancytopenia and hypocellular bone marrow. The pathophysiology is immune mediated in most cases, with activated type 1 cytotoxic T cells implicated. Acquired aplastic anemia can now be cured or ameliorated by stem-cell transplantation or immunosuppressive drug therapy such as antithymocyte globulin or cyclosporine. We present a rare case report of a 68-year old patient with acquired severe aplastic anemia with repeated cerebral infarctions at the beginning of immunosuppressive therapy. He started immunosuppressive drug therapy with antithymocyte globulin and cyclosporine. During follow-up, magnetic resonance imaging revealed high signals at right thalamus and right pons by diffusion-weighted image. He was diagnosed with repeated cerebral infarctions of right thalamus and right pons. We successfully managed cerebral infarctions by frequent transfusions, edaravone administration, keeping the trough of serum cyclosporine (CsA) concentration around lower limit. This is the first report of successful management of acquired aplastic anemia with repeated cerebral infarctions.

Cell Death and Tissue Remodeling in Planarian Regeneration

Developmental Biology. Feb, 2010  |  Pubmed ID: 19766622

Many long-lived organisms, including humans, can regenerate some adult tissues lost to physical injury or disease. Much of the previous research on mechanisms of regeneration has focused on adult stem cells, which give rise to new tissue necessary for the replacement of missing body parts. Here we report that apoptosis of differentiated cells complements stem cell division during regeneration in the planarian Schmidtea mediterranea. Specifically, we developed a whole-mount TUNEL assay that allowed us to document two dramatic increases in the rate of apoptosis following amputation-an initial localized response near the wound site and a subsequent systemic response that varies in magnitude depending on the type of fragment examined. The latter cell death response can be induced in uninjured organs, occurs in the absence of planarian stem cells, and can also be triggered by prolonged starvation. Taken together, our results implicate apoptosis in the restoration of proper anatomical scale and proportion through remodeling of existing tissues. We also report results from initial mechanistic studies of apoptosis in planarians, which revealed that a S. mediterranea homolog of the antiapoptotic gene BCL2 is required for cell survival in adult animals. We propose that apoptosis is a central mechanism working in concert with stem cell division to restore anatomical form and function during metazoan regeneration.

[The Prevalence of Cigarette Smoking Among Asthmatic Adults and Association of Smoking with Emergency Department Visits]

Nihon KokyÅ«ki Gakkai Zasshi = the Journal of the Japanese Respiratory Society. Feb, 2010  |  Pubmed ID: 20184238

We examined the prevalence of cigarette smoking among adults with acute asthma and the relationship between smoking status and visits to the emergency department of a hospital.

Imaging and Biodistribution of Her2/neu Expression in Non-small Cell Lung Cancer Xenografts with Cu-labeled Trastuzumab PET

Cancer Science. Apr, 2010  |  Pubmed ID: 20219072

Non-small cell lung carcinomas (NSCLC) overexpress the Her2/neu gene in approximately 59% of cases. Trastuzumab, a humanized monoclonal antibody, interferes with Her2 signaling and is approved for the treatment of Her2/neu overexpressing breast cancer. However, its therapeutic use in Her2/neu overexpressing NSCLC remains obscure. The present study aimed to determine the role of (64)Cu-labeled trastuzumab positron emission tomography (PET) for non-invasive imaging of Her2/neu expression in NSCLC. Trastuzumab was conjugated with the bifunctional chelator 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) and radiolabeled with (64)Cu. The molecular specificity of DOTA-trastuzumab was determined in NSCLC cell lines with Her2/neu overexpression (NCI-H2170) and negative expression (NCI-H520). Imaging of Her2/neu expression was performed in NCI-H2170 tumor-bearing mice with (64)Cu-DOTA-trastuzumab PET and (64)Cu-DOTA-IgG. In vitro studies revealed specific binding of DOTA-trastuzumab in the Her2/neu positive NCI-H2170 cells, while no binding was seen in the Her2/neu negative NCI-H520 cell line. Biodistribution and PET studies revealed a significantly high accumulation of (64)Cu-DOTA-trastuzumab in the Her2/neu overexpressing NCI-H2170 tumor at 24 and 48 h post-injection (21.4 +/- 1.4% and 23.2 +/- 5.1% injection dose/gram (% ID/g), respectively). PET imaging of Her2/neu negative NCI-H520 tumors showed much less uptake of (64)Cu-DOTA-trastuzumab (4.0% ID/g). The NCI-H2170 tumor uptake of (64)Cu-DOTA-trastuzumab was significantly higher than that of (64)Cu-DOTA-IgG (P < 0.0001). (64)Cu-DOTA-trastuzumab showed a very clear image of a Her2/neu positive tumor and appeared to be effective as a PET tracer for imaging of Her2/neu gene expression in NSCLC, suggesting its potential clinical use for identifying patients that might benefit from trastuzumab-based therapy.

Two Cyclin-dependent Kinase Pathways Are Essential for Polarized Trafficking of Presynaptic Components

Cell. May, 2010  |  Pubmed ID: 20510931

Polarized trafficking of synaptic proteins to axons and dendrites is crucial to neuronal function. Through forward genetic analysis in C. elegans, we identified a cyclin (CYY-1) and a cyclin-dependent Pctaire kinase (PCT-1) necessary for targeting presynaptic components to the axon. Another cyclin-dependent kinase, CDK-5, and its activator p35, act in parallel to and partially redundantly with the CYY-1/PCT-1 pathway. Synaptic vesicles and active zone proteins mostly mislocalize to dendrites in animals defective for both PCT-1 and CDK-5 pathways. Unlike the kinesin-3 motor, unc-104/Kif1a mutant, cyy-1 cdk-5 double mutants have no reduction in anterogradely moving synaptic vesicle precursors (SVPs) as observed by dynamic imaging. Instead, the number of retrogradely moving SVPs is dramatically increased. Furthermore, this mislocalization defect is suppressed by disrupting the retrograde motor, the cytoplasmic dynein complex. Thus, PCT-1 and CDK-5 pathways direct polarized trafficking of presynaptic components by inhibiting dynein-mediated retrograde transport and setting the balance between anterograde and retrograde motors.

Detection of Neuron Membranes in Electron Microscopy Images Using a Serial Neural Network Architecture

Medical Image Analysis. Dec, 2010  |  Pubmed ID: 20598935

Study of nervous systems via the connectome, the map of connectivities of all neurons in that system, is a challenging problem in neuroscience. Towards this goal, neurobiologists are acquiring large electron microscopy datasets. However, the shear volume of these datasets renders manual analysis infeasible. Hence, automated image analysis methods are required for reconstructing the connectome from these very large image collections. Segmentation of neurons in these images, an essential step of the reconstruction pipeline, is challenging because of noise, anisotropic shapes and brightness, and the presence of confounding structures. The method described in this paper uses a series of artificial neural networks (ANNs) in a framework combined with a feature vector that is composed of image intensities sampled over a stencil neighborhood. Several ANNs are applied in series allowing each ANN to use the classification context provided by the previous network to improve detection accuracy. We develop the method of serial ANNs and show that the learned context does improve detection over traditional ANNs. We also demonstrate advantages over previous membrane detection methods. The results are a significant step towards an automated system for the reconstruction of the connectome.

PET Imaging of Norepinephrine Transporter-expressing Tumors Using 76Br-meta-bromobenzylguanidine

Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine. Sep, 2010  |  Pubmed ID: 20720048

Meta-iodobenzylguanidine (MIBG) labeled with (123)I or (131)I has been widely used for the diagnosis and radiotherapy of norepinephrine transporter (NET)-expressing tumors. However, (123)I/(131)I-MIBG has limitations for detecting small lesions because of its lower spatial resolution than PET tracers. In this study, meta-bromobenzylguanidine (MBBG) labeled with (76)Br (half-life, 16.1 h), an attractive positron emitter, was prepared and evaluated as a potential PET tracer for imaging NET-expressing tumors.

Protein Localization in Electron Micrographs Using Fluorescence Nanoscopy

Nature Methods. Jan, 2011  |  Pubmed ID: 21102453

A complete portrait of a cell requires a detailed description of its molecular topography: proteins must be linked to particular organelles. Immunocytochemical electron microscopy can reveal locations of proteins with nanometer resolution but is limited by the quality of fixation, the paucity of antibodies and the inaccessibility of antigens. Here we describe correlative fluorescence electron microscopy for the nanoscopic localization of proteins in electron micrographs. We tagged proteins with the fluorescent proteins Citrine or tdEos and expressed them in Caenorhabditis elegans, fixed the worms and embedded them in plastic. We imaged the tagged proteins from ultrathin sections using stimulated emission depletion (STED) microscopy or photoactivated localization microscopy (PALM). Fluorescence correlated with organelles imaged in electron micrographs from the same sections. We used these methods to localize histones, a mitochondrial protein and a presynaptic dense projection protein in electron micrographs.

Complexin Maintains Vesicles in the Primed State in C. Elegans

Current Biology : CB. Jan, 2011  |  Pubmed ID: 21215631

Complexin binds the SNARE complex at synapses and regulates exocytosis, but genetic studies indicate contradictory roles: in flies it predominantly inhibits synaptic vesicle fusion, whereas in mice it promotes evoked responses.

CYY-1/cyclin Y and CDK-5 Differentially Regulate Synapse Elimination and Formation for Rewiring Neural Circuits

Neuron. May, 2011  |  Pubmed ID: 21609829

The assembly and maturation of neural circuits require a delicate balance between synapse formation and elimination. The cellular and molecular mechanisms that coordinate synaptogenesis and synapse elimination are poorly understood. In C. elegans, DD motoneurons respecify their synaptic connectivity during development by completely eliminating existing synapses and forming new synapses without changing cell morphology. Using loss- and gain-of-function genetic approaches, we demonstrate that CYY-1, a cyclin box-containing protein, drives synapse removal in this process. In addition, cyclin-dependent kinase-5 (CDK-5) facilitates new synapse formation by regulating the transport of synaptic vesicles to the sites of synaptogenesis. Furthermore, we show that coordinated activation of UNC-104/Kinesin3 and Dynein is required for patterning newly formed synapses. During the remodeling process, presynaptic components from eliminated synapses are recycled to new synapses, suggesting that signaling mechanisms and molecular motors link the deconstruction of existing synapses and the assembly of new synapses during structural synaptic plasticity.

[Observation of De Novo Formation and Growth of an Intracranial Aneurysm: a Case Report]

No Shinkei Geka. Neurological Surgery. Jul, 2011  |  Pubmed ID: 21719914

We observed a de novo formation and growth of an aneurysm in a 43-year-old woman who was followed up after treatment of a subarachnoid hemorrhage (SAH). In 2002, the patient, whose mother had a history of SAH, presented with SAH at the age of 36. Three-dimensional computed tomography angiography (3D-CTA) and digital subtraction angiography showed an aneurysm in the right internal carotid-posterior communicating artery. The aneurysm was clipped and postoperative course was uneventful without neurological deficit. The patient was followed up by 3D-CTA and magnetic resonance angiography every 6 months, because of an untreated small aneurysm, 3 mm in diameter, in the left middle cerebral artery (MCA). The MCA aneurysm remained unchanged but a de novo aneurysm, 1.5 mm in diameter, developed in the right anterior cerebral artery (ACA) 6 years after the first surgery. The ACA aneurysm grew to 4 mm in diameter during the following 10 months but the MCA aneurysm remained unchanged. Both aneurysms were clipped in one session. The MCA aneurysm had a smooth wall but the ACA aneurysm had an irregular and thin wall. The postoperative course was uneventful. Young female patients who have developed SAH with familial history, like this case, should receive long-term follow up to check whether a de novo aneurysm has developed.

Preparation and Biological Evaluation of 3-[(76)Br]bromo-α-methyl-L-tyrosine, a Novel Tyrosine Analog for Positron Emission Tomography Imaging of Tumors

Nuclear Medicine and Biology. Aug, 2011  |  Pubmed ID: 21843782

3-[(18)F]fluoro-α-methyl-l-tyrosine ([(18)F]FAMT) is a useful amino acid tracer for positron emission tomography (PET) imaging of malignant tumors. FAMT analogs labeled with (76)Br, a positron emitter with a long half-life (t(1/2)=16.1 h), could potentially be widely used as amino acid tracers for tumor imaging. In this study, 3-[(76)Br]bromo-α-methyl-l-tyrosine ([(76)Br]BAMT) was designed, and its usefulness was evaluated as a novel PET tracer for imaging malignant tumors.

Opposing Activities of LIT-1/NLK and DAF-6/patched-related Direct Sensory Compartment Morphogenesis in C. Elegans

PLoS Biology. Aug, 2011  |  Pubmed ID: 21857800

Glial cells surround neuronal endings to create enclosed compartments required for neuronal function. This architecture is seen at excitatory synapses and at sensory neuron receptive endings. Despite the prevalence and importance of these compartments, how they form is not known. We used the main sensory organ of C. elegans, the amphid, to investigate this issue. daf-6/Patched-related is a glia-expressed gene previously implicated in amphid sensory compartment morphogenesis. By comparing time series of electron-microscopy (EM) reconstructions of wild-type and daf-6 mutant embryos, we show that daf-6 acts to restrict compartment size. From a genetic screen, we found that mutations in the gene lit-1/Nemo-like kinase (NLK) suppress daf-6. EM and genetic studies demonstrate that lit-1 acts within glia, in counterbalance to daf-6, to promote sensory compartment expansion. Although LIT-1 has been shown to regulate Wnt signaling, our genetic studies demonstrate a novel, Wnt-independent role for LIT-1 in sensory compartment size control. The LIT-1 activator MOM-4/TAK1 is also important for compartment morphogenesis and both proteins line the glial sensory compartment. LIT-1 compartment localization is important for its function and requires neuronal signals. Furthermore, the conserved LIT-1 C-terminus is necessary and sufficient for this localization. Two-hybrid and co-immunoprecipitation studies demonstrate that the LIT-1 C-terminus binds both actin and the Wiskott-Aldrich syndrome protein (WASP), an actin regulator. We use fluorescence light microscopy and fluorescence EM methodology to show that actin is highly enriched around the amphid sensory compartment. Finally, our genetic studies demonstrate that WASP is important for compartment expansion and functions in the same pathway as LIT-1. The studies presented here uncover a novel, Wnt-independent role for the conserved Nemo-like kinase LIT-1 in controlling cell morphogenesis in conjunction with the actin cytoskeleton. Our results suggest that the opposing daf-6 and lit-1 glial pathways act together to control sensory compartment size.

Multi-stage Optical FDM of 12-channel 10-Gb/s Data with 20-GHz Exact Channel Spacing Using Fiber Cross-phase Modulation with Optical Subcarrier Signals

Optics Express. Dec, 2011  |  Pubmed ID: 22274033

A sequential optical frequency-division multiplexing technique using cross-phase modulation in fibers with exactly frequency-controlled optical subcarrier signals is proposed and demonstrated. 12 channels of 10-Gb/s ASK/DPSK signals with 20-GHz exact channel spacing are successfully multiplexed all-optically at 12 stages with 1-km intervals.

V-ATPase V1 Sector is Required for Corpse Clearance and Neurotransmission in Caenorhabditis Elegans

Genetics. Jun, 2012  |  Pubmed ID: 22426883

The vacuolar-type ATPase (V-ATPase) is a proton pump composed of two sectors, the cytoplasmic V(1) sector that catalyzes ATP hydrolysis and the transmembrane V(o) sector responsible for proton translocation. The transmembrane V(o) complex directs the complex to different membranes, but also has been proposed to have roles independent of the V(1) sector. However, the roles of the V(1) sector have not been well characterized. In the nematode Caenorhabditis elegans there are two V(1) B-subunit genes; one of them, vha-12, is on the X chromosome, whereas spe-5 is on an autosome. vha-12 is broadly expressed in adults, and homozygotes for a weak allele in vha-12 are viable but are uncoordinated due to decreased neurotransmission. Analysis of a null mutation demonstrates that vha-12 is not required for oogenesis or spermatogenesis in the adult germ line, but it is required maternally for early embryonic development. Zygotic expression begins during embryonic morphogenesis, and homozygous null mutants arrest at the twofold stage. These mutant embryos exhibit a defect in the clearance of apoptotic cell corpses in vha-12 null mutants. These observations indicate that the V(1) sector, in addition to the V(o) sector, is required in exocytic and endocytic pathways.

Semi-Automated Neuron Boundary Detection and Nonbranching Process Segmentation in Electron Microscopy Images

Neuroinformatics. May, 2012  |  Pubmed ID: 22644867

Neuroscientists are developing new imaging techniques and generating large volumes of data in an effort to understand the complex structure of the nervous system. The complexity and size of this data makes human interpretation a labor-intensive task. To aid in the analysis, new segmentation techniques for identifying neurons in these feature rich datasets are required. This paper presents a method for neuron boundary detection and nonbranching process segmentation in electron microscopy images and visualizing them in three dimensions. It combines both automated segmentation techniques with a graphical user interface for correction of mistakes in the automated process. The automated process first uses machine learning and image processing techniques to identify neuron membranes that deliniate the cells in each two-dimensional section. To segment nonbranching processes, the cell regions in each two-dimensional section are connected in 3D using correlation of regions between sections. The combination of this method with a graphical user interface specially designed for this purpose, enables users to quickly segment cellular processes in large volumes.

UNC-41/stonin Functions with AP2 to Recycle Synaptic Vesicles in Caenorhabditis Elegans

PloS One. 2012  |  Pubmed ID: 22808098

The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ∼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin.

Visualizing Proteins in Electron Micrographs at Nanometer Resolution

Methods in Cell Biology. 2012  |  Pubmed ID: 22857934

To understand protein function, we need a detailed description of the molecular topography of the cell. The subcellular localization of proteins can be revealed using genetically encoded fluorescent proteins or immunofluorescence. However, the precise localization of proteins cannot be resolved due to the diffraction limit of light. Recently, the diffraction barrier has been overcome by employing several microscopy techniques. Using super-resolution fluorescence microscopy, one can pinpoint the location of proteins at a resolution of 20 nm or even less. However, the cellular context is often absent in these images. Recently, we developed a method for visualizing the subcellular structures in super-resolution images. Here we describe the method with two technical improvements. First, we optimize the method to preserve more fluorescence without compromising the morphology. Second, we implement ground-state depletion and single-molecule return (GSDIM) imaging, which does not rely on photoactivatable fluorescent proteins. These improvements extend the utility of nano-resolution fluorescence electron microscopy (nano-fEM).

[Retrospective Analysis of Treatment Outcomes in 70 Patients with T(8;21) Acute Myeloid Leukemia]

[Rinshō Ketsueki] The Japanese Journal of Clinical Hematology. Jul, 2012  |  Pubmed ID: 22975772

We conducted a retrospective study to evaluate outcomes and prognostic factors of newly diagnosed patients with t(8;21) acute myeloid leukemia (AML). There were 70 patients (43 men and 27 women) with a median age of 48 years old (range, 17∼76 years old). Sixty-five patients achieved complete remission (CR) after induction chemotherapy. Fifty-seven patients received consolidation chemotherapy based on the policy of not performing allogeneic hematopoietic stem cell transplantation (allo-HSCT) at the time of first CR. Twenty-seven of the 57 patients relapsed (relapse rate, 47%). The median time from the achievement of the first CR to relapse was 307 days (96∼1,256 days). A white blood cell count of more than 25,400/μl at diagnosis was associated with a higher relapse rate than a white blood cell count of less than or equal to 25,400/μl (75% vs. 43%, P=0.04). Nineteen of the 25 relapsed patients who received re-induction therapy experienced a second CR (second CR rate, 76%). Twenty-six patients (5 with first CR, 12 with second CR, and 9 without remission) received allo-HSCT. The five-year overall survival and disease-free survival rates were 61% and 45%, respectively. Patients with t(8;21) AML had a high CR rate, but about half of them relapsed. However, this report could not show prognostic factors for the identification of patients who should receive allo-HSCT at the time of their first CR.

Three-dimensional and Multienergy Gamma-ray Simultaneous Imaging by Using a Si/CdTe Compton Camera

Radiology. Jun, 2013  |  Pubmed ID: 23418002

To develop a silicon (Si) and cadmium telluride (CdTe) imaging Compton camera for biomedical application on the basis of technologies used for astrophysical observation and to test its capacity to perform three-dimensional (3D) imaging.

AP2 Hemicomplexes Contribute Independently to Synaptic Vesicle Endocytosis

ELife. 2013  |  Pubmed ID: 23482940

The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes. DOI:

Synapse Location During Growth Depends on Glia Location

Cell. Jul, 2013  |  Pubmed ID: 23870123

Synaptic contacts are largely established during embryogenesis and are then maintained during growth. To identify molecules involved in this process, we conducted a forward genetic screen in C. elegans and identified cima-1. In cima-1 mutants, synaptic contacts are correctly established during embryogenesis, but ectopic synapses emerge during postdevelopmental growth. cima-1 encodes a solute carrier in the SLC17 family of transporters that includes sialin, a protein that when mutated in humans results in neurological disorders. cima-1 does not function in neurons but rather functions in the nearby epidermal cells to correctly position glia during postlarval growth. Our findings indicate that CIMA-1 antagonizes the FGF receptor (FGFR), and does so most likely by inhibiting FGFR's role in epidermal-glia adhesion rather than signaling. Our data suggest that epidermal-glia crosstalk, in this case mediated by a transporter and the FGF receptor, is vital to preserve embryonically derived circuit architecture during postdevelopmental growth.

Ultrafast Endocytosis at Caenorhabditis Elegans Neuromuscular Junctions

ELife. Sep, 2013  |  Pubmed ID: 24015355

Synaptic vesicles can be released at extremely high rates, which places an extraordinary demand on the recycling machinery. Previous ultrastructural studies of vesicle recycling were conducted in dissected preparations using an intense stimulation to maximize the probability of release. Here, a single light stimulus was applied to motor neurons in intact Caenorhabditis elegans nematodes expressing channelrhodopsin, and the animals rapidly frozen. We found that docked vesicles fuse along a broad active zone in response to a single stimulus, and are replenished with a time constant of about 2 s. Endocytosis occurs within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions. These studies suggest that synaptic vesicle endocytosis may occur on a millisecond time scale following a single physiological stimulus in the intact nervous system and is unlikely to conform to current models of endocytosis. DOI:

Ultrafast Endocytosis at Mouse Hippocampal Synapses

Nature. Dec, 2013  |  Pubmed ID: 24305055

To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated approximately 20 s after fusion by the assembly of clathrin scaffolds or in approximately 1 s by the reversal of fusion pores via 'kiss-and-run' endocytosis. Here we use optogenetics to stimulate cultured hippocampal neurons with a single stimulus, rapidly freeze them after fixed intervals and examine the ultrastructure using electron microscopy--'flash-and-freeze' electron microscopy. Docked vesicles fuse and collapse into the membrane within 30 ms of the stimulus. Compensatory endocytosis occurs within 50 to 100 ms at sites flanking the active zone. Invagination is blocked by inhibition of actin polymerization, and scission is blocked by inhibiting dynamin. Because intact synaptic vesicles are not recovered, this form of recycling is not compatible with kiss-and-run endocytosis; moreover, it is 200-fold faster than clathrin-mediated endocytosis. It is likely that 'ultrafast endocytosis' is specialized to restore the surface area of the membrane rapidly.

Nanometer-resolution Fluorescence Electron Microscopy (nano-EM) in Cultured Cells

Methods in Molecular Biology (Clifton, N.J.). 2014  |  Pubmed ID: 24357377

Nano-resolution fluorescence electron microscopy (nano-fEM) pinpoints the location of individual proteins in electron micrographs. Plastic sections are first imaged using a super-resolution fluorescence microscope and then imaged on an electron microscope. The two images are superimposed to correlate the position of labeled proteins relative to subcellular structures. Here, we describe the method in detail and present five technical advancements: the use of uranyl acetate during the freeze-substitution to enhance the contrast of tissues and reduce the loss of fluorescence, the use of ground-state depletion instead of photoactivation for temporal control of fluorescence, the use of organic fluorophores instead of fluorescent proteins to obtain brighter fluorescence signals, the use of tissue culture cells to broaden the utility of the method, and the use of a transmission electron microscope to achieve sharper images of ultrastructure.

Two Rab2 Interactors Regulate Dense-core Vesicle Maturation

Neuron. Apr, 2014  |  Pubmed ID: 24698274

Peptide neuromodulators are released from a unique organelle: the dense-core vesicle. Dense-core vesicles are generated at the trans-Golgi and then sort cargo during maturation before being secreted. To identify proteins that act in this pathway, we performed a genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We identified two conserved Rab2-binding proteins: RUND-1, a RUN domain protein, and CCCP-1, a coiled-coil protein. RUND-1 and CCCP-1 colocalize with RAB-2 at the Golgi, and rab-2, rund-1, and cccp-1 mutants have similar defects in sorting soluble and transmembrane dense-core vesicle cargos. RUND-1 also interacts with the Rab2 GAP protein TBC-8 and the BAR domain protein RIC-19, a RAB-2 effector. In summary, a pathway of conserved proteins controls the maturation of dense-core vesicles at the trans-Golgi network.

Identification of a Novel SEPT9-ABL1 Fusion Gene in a Patient with T-cell Prolymphocytic Leukemia

Leukemia Research Reports. 2014  |  Pubmed ID: 25068103

T-cell prolymphocytic leukemia (T-PLL), a rare type of peripheral T-cell leukemia, is characterized by marked splenomegaly with rapidly progressive lymphocytosis and a poor prognosis. Nine kinds of ABL1 chimeric genes have been identified in various kinds of hematological malignancies, such as chronic myeloid leukemia and B- or T-lymphoblastic leukemia. However, there have been no reports describing T-PLL cases with ABL1 rearrangements. We herein report a case of T-PLL with a novel SEPT9-ABL1 fusion gene which induced strong resistance to tyrosine kinase inhibitors such as imatinib and dasatinib.

Clathrin Regenerates Synaptic Vesicles from Endosomes

Nature. Nov, 2014  |  Pubmed ID: 25296249

Ultrafast endocytosis can retrieve a single, large endocytic vesicle as fast as 50-100 ms after synaptic vesicle fusion. However, the fate of the large endocytic vesicles is not known. Here we demonstrate that these vesicles transition to a synaptic endosome about one second after stimulation. The endosome is resolved into coated vesicles after 3 s, which in turn become small-diameter synaptic vesicles 5-6 s after stimulation. We disrupted clathrin function using RNA interference (RNAi) and found that clathrin is not required for ultrafast endocytosis but is required to generate synaptic vesicles from the endosome. Ultrafast endocytosis fails when actin polymerization is disrupted, or when neurons are stimulated at room temperature instead of physiological temperature. In the absence of ultrafast endocytosis, synaptic vesicles are retrieved directly from the plasma membrane by clathrin-mediated endocytosis. These results may explain discrepancies among published experiments concerning the role of clathrin in synaptic vesicle endocytosis.

An Acceptable Incidence of Infusion Site Reactions After Subcutaneous Bortezomib Administration in the Upper Arm in Japanese Patients with Multiple Myeloma

Acta Haematologica. 2015  |  Pubmed ID: 24993672

Development of a Widely Usable Amino Acid Tracer: ⁷⁶Br-α-Methyl-Phenylalanine for Tumor PET Imaging

Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine. May, 2015  |  Pubmed ID: 25814518

Radiolabeled amino acids are superior PET tracers for the imaging of malignant tumors, and amino acids labeled with (76)Br, an attractive positron emitter because of its relatively long half-life (16.2 h), could potentially be a widely usable tumor imaging tracer. In this study, in consideration of its stability and tumor specificity, we designed two (76)Br-labeled amino acid derivatives, 2-(76)Br-bromo-α-methyl-l-phenylalanine (2-(76)Br-BAMP) and 4-(76)Br-bromo-α-methyl-l-phenylalanine (4-(76)Br-BAMP), and investigated their potential as tumor imaging agents.

Preparation and Evaluation of an Astatine-211-labeled Sigma Receptor Ligand for Alpha Radionuclide Therapy

Nuclear Medicine and Biology. Nov, 2015  |  Pubmed ID: 26235266

Sigma receptors are overexpressed in a variety of human tumors, making them potential targets for radionuclide receptor therapy. We have previously synthesized and evaluated (131)I-labeled (+)-2-[4-(4-iodophenyl)piperidino]cyclohexanol [(+)-[(131)I]pIV], which has a high affinity for sigma receptors. Therefore, (+)-[(131)I]pIV significantly inhibited tumor cell proliferation in tumor-bearing mice. In the present study, we report the synthesis and the in vitro and in vivo characterization of (+)-[(211)At]pAtV, an (211)At-labeled sigma receptor ligand, that has potential use in alpha-radionuclide receptor therapy.

Slow or Fast? A Tale of Synaptic Vesicle Recycling

Science (New York, N.Y.). Oct, 2015  |  Pubmed ID: 26430111

Simultaneous Analysis of Silicon and Boron Dissolved in Water by Combination of Electrodialytic Salt Removal and Ion-exclusion Chromatography with Corona Charged Aerosol Detection

Journal of Chromatography. A. Jan, 2016  |  Pubmed ID: 26755416

Selective separation and sensitive detection of dissolved silicon and boron (DSi and DB) in aqueous solution was achieved by combining an electrodialytic ion isolation device (EID) as a salt remover, an ion-exclusion chromatography (IEC) column, and a corona charged aerosol detector (CCAD) in sequence. DSi and DB were separated by IEC on the H(+)-form of a cation exchange resin column using pure water eluent. DSi and DB were detected after IEC separation by the CCAD with much greater sensitivity than by conductimetric detection. The five-channel EID, which consisted of anion and cation acceptors, cathode and anode isolators, and a sample channel, removed salt from the sample prior to the IEC-CCAD. DSi and DB were scarcely attracted to the anion accepter in the EID and passed almost quantitatively through the sample channel. Thus, the coupled EID-IEC-CCAD device can isolate DSi and DB from artificial seawater and hot spring water by efficiently removing high concentrations of Cl(-) and SO4(2-) (e.g., 98% and 80% at 0.10molL(-1) each, respectively). The detection limits at a signal-to-noise ratio of 3 were 0.52μmolL(-1) for DSi and 7.1μmolL(-1) for DB. The relative standard deviations (RSD, n=5) of peak areas were 0.12% for DSi and 4.3% for DB.

Flash-and-Freeze: Coordinating Optogenetic Stimulation with Rapid Freezing to Visualize Membrane Dynamics at Synapses with Millisecond Resolution

Frontiers in Synaptic Neuroscience. 2016  |  Pubmed ID: 27594835

Electron microscopy depicts subcellular structures at synapses exquisitely but only captures static images. To visualize membrane dynamics, we have developed a novel technique, called flash-and-freeze, which induces neuronal activity with a flash of light and captures the membrane dynamics by rapid freezing. For characterizing membrane movements during synaptic transmission, a light-sensitive cation channel, channelrhodopsin, is heterologously expressed in mouse hippocampal neurons or in Caenorhabditis elegans motor neurons. A brief pulse of blue light activates channelrhodopsin and induces an action potential, leading to synaptic transmission. Following the light stimulation, neurons are frozen at different time intervals ranging from 10 ms to 20 s. Electron micrographs are then acquired from each time point to visualize the morphological changes. Using this approach, we have characterized a novel form of endocytosis, ultrafast endocytosis, which rapidly removes excess membrane added to the surface during neurotransmission. The flash-and-freeze approach can be adapted to study other cellular phenomena that can be induced by light-sensitive genetic or pharmacological tools.

The NCA-1 and NCA-2 Ion Channels Function Downstream of Gq and Rho To Regulate Locomotion in Caenorhabditis Elegans

Genetics. Mar, 2017  |  Pubmed ID: 28325749

The heterotrimeric G protein Gq positively regulates neuronal activity and synaptic transmission. Previously, the Rho guanine nucleotide exchange factor Trio was identified as a direct effector of Gq that acts in parallel to the canonical Gq effector phospholipase C. Here we examine how Trio and Rho act to stimulate neuronal activity downstream of Gq in the nematode Caenorhabditis elegans Through two forward genetic screens, we identify the cation channels NCA-1 and NCA-2, orthologs of mammalian NALCN, as downstream targets of the Gq/Rho pathway. By performing genetic epistasis analysis using dominant activating mutations and recessive loss-of-function mutations in the members of this pathway, we show that NCA-1 and NCA-2 act downstream of Gq in a linear pathway. Through cell-specific rescue experiments, we show that function of these channels in head acetylcholine neurons is sufficient for normal locomotion in C. elegans Our results suggest that NCA-1 and NCA-2 are physiologically relevant targets of neuronal Gq-Rho signaling in C. elegans.

Fast and Ultrafast Endocytosis

Current Opinion in Cell Biology. Apr, 2017  |  Pubmed ID: 28391090

Clathrin-mediated endocytosis (CME) is the main endocytic pathway supporting housekeeping functions in cells. However, CME may be too slow to internalize proteins from the cell surface during certain physiological processes such as reaction to stress hormones ('fight-or-flight' reaction), chemotaxis or compensatory endocytosis following exocytosis of synaptic vesicles or hormone-containing vesicles. These processes take place on a millisecond to second timescale and thus require very rapid cellular reaction to prevent overstimulation or exhaustion of the response. There are several fast endocytic processes identified so far: macropinocytosis, activity-dependent bulk endocytosis (ABDE), fast-endophilin-mediated endocytosis (FEME), kiss-and-run and ultrafast endocytosis. All are clathrin-independent and are not constitutively active but may use different molecular mechanisms to rapidly remove receptors and proteins from the cell surface. Here, we review our current understanding of fast and ultrafast endocytosis, their functions, and molecular mechanisms.

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