Studying the O-GlcNAcome of human placentas using banked tissue samples.

O-GlcNAcylation is a dynamic modulator of signaling pathways, equal in magnitude to the widely studied phosphorylation. With the rapid development of tools for its detection at the single protein level, the O-GlcNAc modification rapidly emerged as a novel diagnostic and therapeutic target in human diseases. Yet, mapping the human O-GlcNAcome in various tissues is essential for generating relevant biomarkers. In this study, we used human banked tissue as a sample source to identify O-GlcNAcylated protein targets relevant to human diseases. Using human term placentas, we propose (1) a method to clean frozen banked tissue of blood proteins; (2) an optimized protocol for the enrichment of O-GlcNAcylated proteins using immunoaffinity purification; and (3) a bioinformatic workflow to identify the most promising O-GlcNAc targets. As a proof-of-concept, we used 45 mg of banked placental samples from two pregnancies to generate intracellular protein extracts depleted of blood protein. Then, antibody-based O-GlcNAc enrichment on denatured samples yielded over 2000 unique HexNAc PSMs and 900 unique sites using 300 µg of protein lysate. Due to efficient sample cleanup, we also captured 82 HexNAc proteins with high placental expression. Finally, we provide a bioinformatic tool (CytOVS) to sort the HexNAc proteins based on their cellular localization and extract the most promising O-GlcNAc targets to explore further. To conclude, we provide a simple 3-step workflow to generate a manageable list of O-GlcNAc proteins from human tissue and improve our understanding of O-GlcNAcylation's role in health and diseases.

Phytochrome B regulates Heading date 1 (Hd1)-mediated expression of rice florigen Hd3a and critical day length in rice.

Many plants require circadian clock and light information for the photoperiodic control of flowering. In Arabidopsis, a long-day plant (LDP), flowering is triggered by the circadian clock-controlled expression of CONSTANS (CO) and light stabilization of the CO protein to induce FT (FLOWERING LOCUS T). In rice, a short-day plant (SDP), the CO ortholog Heading date 1 (Hd1) regulates FT ortholog Hd3a, but regulation of Hd3a by Hd1 differs from that in Arabidopsis. Here, we report that phytochrome B (phyB)-mediated suppression of Hd3a is a primary cause of long-day suppression of flowering in rice, based on the three complementary discoveries. First, overexpression of Hd1 causes a delay in flowering under SD conditions and this effect requires phyB, suggesting that light modulates Hd1 control of Hd3a transcription. Second, a single extension of day length decreases Hd3a expression proportionately with the length of daylight. Third, Hd1 protein levels in Hd1-overexpressing plants are not altered in the presence of light. These results also suggest that phyB-mediated suppression of Hd3a expression is a component of the molecular mechanism for critical day length in rice.

Production of a rat monoclonal antibody specific for Myf5.

Myogenic regulatory factors (MRFs) are transcription factors that possess a characteristic basic helix-loop-helix domain. Myf5, MyoD, MRF4, and myogenin are well-known MRF family members that activate muscle-specific genes during differentiation. Myf5 is expressed first among MRFs at the very early phase and plays an important role in myoblast specificity and cell proliferation. Myf5 shares high homology with MyoD, and therefore some commercial Myf5 antibodies are cross-reactive for Myf5 and MyoD. To allow for detailed studies of the function of Myf5, we generated a monoclonal antibody specific for Myf5 utilizing a rat medial iliac lymph node method. Immunoblot analysis using our monoclonal antibody enabled us to identify Myf5 protein from rat myoblast L6E9 cell extract. Moreover, cell immunostaining revealed the nuclear localization of Myf5 in the L6E9 cells. This monoclonal antibody against Myf5 will allow us to perform further detailed studies of Myf5 and Myf5 function.

Generation of a rat monoclonal antibody specific for Pax7.

Pax7 is a nuclear localization protein, well known as a member of the paired box family. It is expressed at a very early stage of muscle differentiation and is also found in muscle satellite cells that are recognized as muscle stem cells. Pax7 is also recognized as a tumor cell marker since it is greatly expressed in various types of tumor cells. Pax7 has homology among other paired family members and is not easy to distinguish one from the others. In this study, we report on the establishment of monoclonal antibodies (MAb) against Pax7 using a rat medial iliac lymph node method. The quality of the antibody was examined by immunoblotting analysis. It was confirmed that the antibody can specifically recognize the Pax7 protein. It was also revealed that the MAb antibody successfully recognizes the nuclear localized Pax7 protein in Ewing's sarcoma cells by immunocytochemistry. The antibody can clearly show the regions of euchromatin and heterochromatin where hoechst is positive.

Neutrino mass, dark matter, and Baryon asymmetry via TeV-scale physics without fine-tuning.

We propose an extended version of the standard model, in which neutrino oscillation, dark matter, and the baryon asymmetry of the Universe can be simultaneously explained by the TeV-scale physics without assuming a large hierarchy among the mass scales. Tiny neutrino masses are generated at the three-loop level due to the exact Z2 symmetry, by which the stability of the dark matter candidate is guaranteed. The extra Higgs doublet is required not only for the tiny neutrino masses but also for successful electroweak baryogenesis. The model provides discriminative predictions especially in Higgs phenomenology, so that it is testable at current and future collider experiments.

[Assessment of pathophysiology based on the left ventricular shape in five patients with midventricular obstructive hypertrophic cardiomyopathy].

OBJECTIVES: The pathophysiology of midventricular obstructive hypertrophic cardiomyopathy (MVO) is unknown. Patients with MVO and MVO-like cardiomyopathy were classified into three groups based on the cardioimaging morphological characteristics of the left ventricle to investigate their complications and treatment. METHODS: Four patients with MVO and one patient with disease-like MVO were admitted in our hospital from 1999 to 2005. Group A consisted of one patient with indications of pressure gradient at mid-ventricle without apical aneurysm, Group B consisted of three patients with indications of pressure gradient and apical aneurysm, and Group C consisted of one patient with hour-glass appearance with apical aneurysm and decreased left ventricular systolic function without pressure gradient. RESULTS: The diagnosis was established during examination for sustained ventricular tachycardia (SVT, three patients), paroxysmal atrial fibrillation (one patient), and coronary artery disease (one patient). Cardiogenic embolization was observed in all cases which originated from atrial fibrillation (one case) and apical aneurysm (two cases). No embolic event occurred in any patient after warfarin therapy. SVT occurred in patients in Groups B and C. SVT refractory to beta-blocker and mexiletine was treated by amiodarone. Apical aneurysmectomy and cryoablation could prevent recurrent SVT with drug resistance. CONCLUSIONS: Four of the five patients with MVO had arrhythmia (atrial fibrillation, SVT) and three had cardiogenic embolization. MVO could be classified into three groups depending on the morphological characteristics and complications. Treatment of MVO should be based on these characteristics.

Effectiveness of paroxetine in the treatment of obsessive-compulsive disorders.

Clomipramine ushered in a new age of pharmacotherapy for obsessive-compulsive disorders, and it also facilitated our understanding of the biological aspects of obsessive-compulsive disorder, focusing on the serotonergic systems. The introduction of selective serotonin reuptake inhibitors has led to great progress in the pharmacological study of obsessive-compulsive disorder based on the serotonin hypothesis. Currently, selective serotonin reuptake inhibitors are positioned as a first-line drug of obsessive-compulsive disorder pharmacotherapy in the various guidelines and algorithms. Among six different selective serotonin reuptake inhibitors (paroxetine, sertraline, fluoxetine, fluvoxamine, citalopram, escitalopram) that are available worldwide, paroxetine has the broadest treatment spectrum and promises great benefits not only for obsessive-compulsive disorder patients, but also for those with comorbid depression and/or various kinds of anxiety disorders. This paper presents several clinical trials of paroxetine carried out, and discusses and reviews the therapeutic strategies for obsessive-compulsive disorder.

Intestinal water absorption through aquaporin 1 expressed in the apical membrane of mucosal epithelial cells in seawater-adapted Japanese eel.

To elucidate the mechanisms associated with water absorption in the intestine, we compared drinking and intestinal water absorption in freshwater- and seawater-adapted Japanese eels, and investigated a possible involvement of aquaporin (AQP) in the absorption of water in the intestine. Seawater eels ingested more water than freshwater eels, the drinking rate being 0.02 ml kg(-1) h(-1) in fresh water and 0.82 ml kg(-1) h(-1) in sea water. In intestinal sacs prepared from freshwater and seawater eels, water absorption increased in time- and hydrostatic pressure-dependent manners. The water absorption rates were greater in seawater sacs than in freshwater sacs, and also greater in the posterior intestine than in the anterior. In view of the enhanced water permeability in the intestine of seawater eel, we cloned two cDNAs encoding AQP from the seawater eel intestine, and identified two eel homologues (S-AQP and L-AQP) of mammalian AQP1. S-AQP and L-AQP possessed the same amino acid sequence, except that one amino acid was lacking in S-AQP and two amino acids were substituted. Eel AQP1 was expressed predominantly in the intestine, and the expression levels were higher in seawater eel than in freshwater eel. Immunocytochemical studies revealed intense AQP1 immunoreaction in the apical surface of columnar epithelial cells in seawater eel, in which the immunoreaction was stronger in the posterior intestine than in the anterior. In contrast, the immunoreaction was faint in the freshwater eel intestine. Preferential localization of AQP1 in the apical membrane of epithelial cells in the posterior intestine of seawater eel indicates that this region of the intestine is responsible for water absorption, and that AQP1 may act as a water entry site in the epithelial cells.