Effect of Yokukansan on sleep disturbance and neuropathic pain in chronic constriction injury using a rat model

Sleep and Biological Rhythms - Patients with chronic pain develop peripheral neuropathy and experience sleep disturbance. Yokukansan is used to treat insomnia and control neuropathic pain. We...     

Detection of eggs in the living white grub beetle Dasylepida ishigakiensis (Coleoptera: Scarabaeidae) by magnetic resonance imaging

The use of magnetic resonance imaging (MRI) as a tool for in vivo detection of eggs in living Dasylepida ishigakiensis Niijima et Kinoshita, a major pest of sugarcane, was explored using females with an ovary at different developmental stages. MRI measurements of beetles were performed at 13 °C to avoid motion artifacts on the MR images. Spin–lattice relaxation time-weighted images allowed the observation of eggs at short acquisition times (2 min, 8 s). By comparing MR images with dissection data, criteria for determining mature eggs in MR images were a clear circular or ellipsoidal shape surrounded by a relatively bright rim and a size typically larger than 1.3 mm in the minor axis. Although small oocytes could not be detected, females with a developed or undeveloped ovary could be clearly distinguished based on MR images. The possibility of confusing the digestive tract as eggs in a female with a less developed ovary can be eliminated using a proton density weighted image.     

Structural basis for regulation of poly‐SUMO chain by a SUMO‐like domain of Nip45

Post‐translational modification by small ubiquitin‐like modifier (SUMO) provides an important regulatory mechanism in diverse cellular processes. Modification of SUMO has been shown to target proteins involved in systems ranging from DNA repair pathways to the ubiquitin‐proteasome degradation system by the action of SUMO‐targeted ubiquitin ligases (STUbLs). STUbLs recognize target proteins modified with a poly‐SUMO chain through their SUMO‐interacting motifs (SIMs). STUbLs are also associated with RENi family proteins, which commonly have two SUMO‐like domains (SLD1 and SLD2) at their C terminus. We have determined the crystal structures of SLD2 of mouse RENi protein, Nip45, in a free form and in complex with a mouse E2 sumoylation enzyme, Ubc9. While Nip45 SLD2 shares a β‐grasp fold with SUMO, the SIM interaction surface conserved in SUMO paralogues does not exist in SLD2. Biochemical data indicates that neither tandem SLDs or SLD2 of Nip45 bind to either tandem SIMs from either mouse STUbL, RNF4 or to those from SUMO‐binding proteins, whose interactions with SUMO have been well characterized. On the other hand, Nip45 SLD2 binds to Ubc9 in an almost identical manner to that of SUMO and thereby inhibits elongation of poly‐SUMO chains. This finding highlights a possible role of the RENi proteins in the modulation of Ubc9‐mediated poly‐SUMO formation. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Towards dynamic metabolic network measurements by multi-dimensional NMR-based fluxomics

Novel technologies for measuring biological systems and methods for visualizing data have led to a revolution in the life sciences. Nuclear magnetic resonance (NMR) techniques can provide information on metabolite structure and metabolic dynamics at the atomic level. We have been developing a new method for measuring the dynamic metabolic network of crude extracts that combines [(13)C(6)]glucose stable isotope labeling of Arabidopsis thaliana and multi-dimensional heteronuclear NMR analysis, whereas most conventional metabolic flux analyses examine proteinogenic amino acids that are specifically labeled with partially labeled substrates such as [2-(13)C(1)]glucose or 10% [(13)C(6)]glucose. To show the validity of our method, we investigated how to obtain information about biochemical reactions, C-C bond formation, and the cleavage of the main metabolites, such as free amino acids, in crude extracts based on the analysis of the (13)C-(13)C coupling pattern in 2D-NMR spectra. For example, the combination of different extraction solvents allows one to distinguish complicated (13)C-(13)C fine couplings at the C2 position of amino acids. As another approach, f1-f3 projection of the HCACO spectrum also helps in the analysis of (13)C-(13)C connectivities. Using these new methods, we present an example that involves monitoring the incorporation profile of [(13)C(6)]glucose into A. thaliana and its metabolic dynamics, which change in a time-dependent manner with atmospheric (12)CO(2) assimilation.     

In-depth proteomic profiling of the normal human kidney glomerulus using two-dimensional protein prefractionation in combination with liquid chromatography-tandem mass spectrometry

The kidney glomerulus plays a pivotal role in ultrafiltration of plasma into urine and also is the locus of kidney disease progressing to chronic renal failure. We have focused proteomic analysis on the glomerulus that is most proximal to the disease locus. In the present study, we aimed to provide a confident, in-depth profiling of the glomerulus proteome. The glomeruli were highly purified from the kidney cortex from a male, 68-year-old patient who underwent nephroureterectomy due to ureter carcinoma. The patient was normal in clinical examinations including serum creatinine and urea levels and liver function, and did not receive any chemotherapy and radiotherapy. The cortical tissue was histologically normal, and no significant deposition of immunoglobulins and complement C3 was observed. We employed a novel strategy of protein separation using 1D (SDS-PAGE) and 2D (solution-phase IEF in combination with SDS-PAGE) prefractionation prior to the shotgun analysis with LC-MS/MS. The protein prefractionation produced 90 fractions, and eventually provided a confident set of identified proteins consisting of 6686 unique proteins (3679 proteins with two or more peptide matches and 3007 proteins with one peptide match), representing 2966 distinct genes. All the identified proteins were annotated and classified in terms of molecular function and biological process, compiled into 1D and 2D protein arrays, consisting of 15 and 75 sections, corresponding to the protein fractions which were defined by MW and pI range, and deposited on a Web-based database (http://www.hkupp.org). The most remarkable feature of the glomerulus proteome was a high incidence of identification of cytoskeleton-related proteins, presumably reflecting the well-developed, cytoskeletal organization of glomerular cells related to their physiological functions.     

Reveromycin A biosynthesis uses RevG and RevJ for stereospecific spiroacetal formation

Spiroacetal compounds are ubiquitous in nature, and their stereospecific structures are responsible for diverse pharmaceutical activities. Elucidation of the biosynthetic mechanisms that are involved in spiroacetal formation will open the door to efficient generation of stereospecific structures that are otherwise hard to synthesize chemically. However, the biosynthesis of these compounds is poorly understood, owing to difficulties in identifying the responsible enzymes and analyzing unstable intermediates. Here we comprehensively describe the spiroacetal formation involved in the biosynthesis of reveromycin A, which inhibits bone resorption and bone metastases of tumor cells by inducing apoptosis in osteoclasts. We performed gene disruption, systematic metabolite analysis, feeding of labeled precursors and conversion studies with recombinant enzymes. We identified two key enzymes, dihydroxy ketone synthase and spiroacetal synthase, and showed in vitro reconstruction of the stereospecific spiroacetal structure from a stable acyclic precursor. Our findings provide insights into the creation of a variety of biologically active spiroacetal compounds for drug leads.     

Adaptation to left-right reversed vision rapidly activates ipsilateral visual cortex in humans.

The brain mechanisms of adaptation to visual transposition are of increasing interest, not only for research on sensory-motor coordination, but also for neuropsychological rehabilitation. Sugita [Nature 380 (1996) 523] found that after adaptation to left-right reversed vision for one and a half months, monkey V1 neurons responded to stimuli presented not only in the contralateral visual field, but also in the ipsilateral visual field. To identify the underlying neuronal mechanisms of adaptation to visual transposition, we conducted fMRI and behavioral experiments for which four adult human subjects wore left-right reversing goggles for 35/39 days, and investigated: (1) whether ipsilateral V1 activation can be induced in human adult subjects; (2) if yes, when the ipsilateral activity starts, and what kind of behavioral/psychological changes occur accompanying the ipsilateral activity; (3) whether other visual cortices also show an ipsilateral activity change. The results of behavioral experiments showed that visuomotor coordinative function and internal representation of peripersonal space rapidly adapted to the left-right reversed vision within the first or second week. Accompanying these behavioral changes, we found that both primary (V1) and extrastriate (MT/MST) visual cortex in human adults responded to visual stimuli presented in the ipsilateral visual field. In addition, the ipsilateral activity started much sooner than the one and a half months, which had been expected from the monkey neurophysiological study. The results of the present study serve as physiological evidence of large-scale, cross-hemisphere, cerebral plasticity that exists even in adult human brain.     

Abnormalities in aggression and anxiety in transgenic mice overexpressing activin E

To study the function of activin E, a TGF-β superfamily member, in the regulation of affective behavior, we investigated the behavior of transgenic mice overexpressing activin E (TgActβE mice). Male TgActβE mice showed aggressive behavior in resident-intruder tests. In elevated plus-maze tests, the percentage of open arm entries was significantly increased in female TgActβE mice compared with that in wild-type mice. Furthermore, female TgActβE mice stayed in the central area for a significantly longer time than wild-type mice in open field tests. These results indicated that TgActβE mice had less anxiety-like behavior. The number of restraint-stress-evoked c-Fos-positive cells in the hypothalamic paraventricular nucleus in TgActβE mice was significantly decreased compared with that in wild-type mice. This suggests that synthesis of corticotrophin-releasing hormone induced by stress was decreased in TgActβE mice. Taking these results together, activin E may act as a regulator of the hypothalamic-pituitary-adrenal axis.     

Physiological stress exacerbates murine colitis by enhancing proinflammatory cytokine expression that is dependent on IL-18

Psychological stress is an environmental factor considered to be a precipitating factor of inflammatory bowel disease. Interleukin (IL)-18 plays a role in stress-induced aggravation in some diseases. The aim of this study was to establish a model of murine colitis exacerbated by psychological stress and to clarify the role of IL-18 in this model. Male C57Bl/6 mice and IL-18(-/-) mice were used for this study. The mice received dextran sulfate sodium (DSS) for induction of colitis. Some mice were exposed to psychological stress using a communication box. Body weight, colonic length, and histological inflammation were measured for assessment of colitis. Tumor necrosis factor (TNF)-α and IL-18 expression in the colon and IL-18 expression in the adrenal gland were analyzed using real-time PCR. The effect of anti-IL-18 antibody was also investigated. Effects of TNF-α and IL-18 on cytokine expressions were studied using the colonic epithelial cell line LS174T. Induction of psychological stress in DSS-treated wild-type mice significantly exacerbated colitis with enhanced expression of proinflammatory cytokines and IL-18. However, induction of psychological stress in DSS-treated IL-18(-/-) mice did not aggravate colitis compared with that in the IL-18(-/-) group given only DSS treatment. Stress-induced aggravation of colitis was ameliorated significantly by anti-IL-18 antibody treatment. IL-18 did not enhance TNF-α-induced expression of intercellular adhesion molecule-1 or IL-8 in LS174T. We established a model of colitis exacerbated by psychological stress. Psychological stress enhanced IL-18 expression and plays a proinflammatory role in stress-induced aggravation of colitis.