Novel mutations affecting axon guidance in zebrafish and a role for plexin signalling in the guidance of trigeminal and facial nerve axons

In zebrafish embryos, the axons of the posterior trigeminal (Vp) and facial (VII) motoneurons project stereotypically to a small number of target muscles derived from the first and second branchial arches (BA1, BA2). Use of the Islet1 (Isl1)-GFP transgenic line enabled precise real-time observations of the growth cone behaviour of the Vp and VII motoneurons within BA1 and BA2. Screening for N-ethyl-N-nitrosourea-induced mutants identified seven distinct mutations affecting different steps in the axonal pathfinding of these motoneurons. The class 1 mutations caused severe defasciculation and abnormal pathfinding in both Vp and VII motor axons before they reached their target muscles in BA1. The class 2 mutations caused impaired axonal outgrowth of the Vp motoneurons at the BA1-BA2 boundary. The class 3 mutation caused impaired axonal outgrowth of the Vp motoneurons within the target muscles derived from BA1 and BA2. The class 4 mutation caused retraction of the Vp motor axons in BA1 and abnormal invasion of the VII motor axons in BA1 beyond the BA1-BA2 boundary. Time-lapse observations of the class 1 mutant, vermicelli (vmc), which has a defect in the plexin A3 (plxna3) gene, revealed that Plxna3 acts with its ligand Sema3a1 for fasciculation and correct target selection of the Vp and VII motor axons after separation from the common pathways shared with the sensory axons in BA1 and BA2, and for the proper exit and outgrowth of the axons of the primary motoneurons from the spinal cord.     

Structural Basis of Pharmacological Chaperoning for Human β-Galactosidase

G_M1 gangliosidosis and Morquio B disease are autosomal recessive diseases caused by the defect in the lysosomal β-galactosidase (β-Gal), frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation. Pharmacological chaperone (PC) therapy is a newly developed molecular therapeutic approach by using small molecule ligands of the mutant enzyme that are able to promote the correct folding and prevent endoplasmic reticulum-associated degradation and promote trafficking to the lysosome. In this report, we describe the enzymological properties of purified recombinant human β-Gal^WT and two representative mutations in G_M1 gangliosidosis Japanese patients, β-Gal^R201C and β-Gal^I51T. We have also evaluated the PC effect of two competitive inhibitors of β-Gal. Moreover, we provide a detailed atomic view of the recognition mechanism of these compounds in comparison with two structurally related analogues. All compounds bind to the active site of β-Gal with the sugar-mimicking moiety making hydrogen bonds to active site residues. Moreover, the binding affinity, the enzyme selectivity, and the PC potential are strongly affected by the mono- or bicyclic structure of the core as well as the orientation, nature, and length of the exocyclic substituent. These results provide understanding on the mechanism of action of β-Gal selective chaperoning by newly developed PC compounds.     

Erythropoietin affords additional cardioprotection to preconditioned hearts by enhanced phosphorylation of glycogen synthase kinase-3 beta

The aim of this study was to determine whether erythropoietin (EPO) affords additional cardioprotection to the preconditioned myocardium by enhanced phosphorylation of Akt, STAT3, or glycogen synthase kinase-3beta (GSK-3 beta). Preconditioning (PC) with 5-min ischemia/5-min reperfusion and EPO (5,000 U/kg iv) reduced infarct size (as % of area at risk, %IS/AR) after 20-min ischemia in rat hearts in situ from 56.5 +/- 1.8% to 25.2 +/- 2.1% and to 36.2 +/- 2.8%, respectively. PC-induced protection was significantly inhibited by a protein kinase C inhibitor, chelerythrine (5 mg/kg), and slightly blunted by a phosphatidylinositol-3-kinase inhibitor, wortmannin (15 microg/kg). The opposite pattern of inhibition was observed for EPO-induced protection. The combination of PC and EPO further reduced %IS/AR to 8.9 +/- 1.9%, and this protection was inhibited by chelerythrine and wortmannin. The additive effects of PC and EPO on infarct size were mirrored by their effects on the level of phosphorylated GSK-3 beta at 5 min after reperfusion but not their effects on the level of phospho-Akt or phospho-STAT3. To mimic phosphorylation-induced inhibition of GSK-3 beta activity, SB-216763 (SB), a GSK-3 beta inhibitor, was administered before ischemia or 5 min before reperfusion. Infarct size was significantly reduced by preischemic injection (%IS/AR = 40.4 +/- 2.2% by 0.6 mg/kg SB and 34.0 +/- 1.8% by 1.2 mg/kg SB) and also by prereperfusion injection (%IS/AR = 32.0 +/- 2.0% by 1.2 mg/kg SB). These results suggest that EPO and PC afford additive infarct size-limiting effects by additive phosphorylation of GSK-3beta at the time of reperfusion by Akt-dependent and -independent mechanisms.     

Effects of triiodothyronine on turnover rate and metabolizing enzymes for thyroxine in thyroidectomized rats

Aim

Previous studies in rats have indicated that surgical thyroidectomy represses turnover of serum thyroxine (T₄). However, the mechanism of this process has not been identified. To clarify the mechanism, we studied adaptive variation of metabolic enzymes involved in T₄ turnover.

Main methods

We compared serum T₄ turnover rates in thyroidectomized (Tx) rats with or without infusion of active thyroid hormone, triiodothyronine (T₃). Furthermore, the levels of mRNA expression and activity of the metabolizing enzymes, deiodinase type 1 (D1), type 2 (D2), uridine diphosphate-glucuronosyltransferase (UGT), and sulfotransferase were also compared in several tissues with or without T₃ infusion.

Key findings

After the T₃ infusion, the turnover rate of serum T₄ in Tx rats returned to normal. Although mRNA expression and activity of D1 decreased significantly in both liver and kidneys without T₃ infusion, D2 expression and activity increased markedly in the brain, brown adipose tissue, and skeletal muscle. Surprisingly, hepatic UGT mRNA expression and activity in Tx rats increased significantly in comparison with normal rats, and returned to normal after T₃ infusion.

Significance

This study suggests that repression of the disappearance of serum T₄ in rats after Tx is a homeostatic response to decreased serum T₃ concentrations. Additionally, T₄ glucuronide is a storage form of T₄, but may also have biological significance. These results suggest strongly that repression of deiodination of T₄ by D1 in the liver and kidneys plays a major role in thyroid hormone homeostasis in Tx rats, and that hepatic UGT also plays a key role in this mechanism.     

Short term exposure to 1439 MHz pulsed TDMA field does not alter melatonin synthesis in rats

The widespread use of the mobile phone has initiated many studies on the possible adverse effects of a high frequency electromagnetic field (EMF), which is used in mobile phones. A low frequency EMF is reported to suppress melatonin synthesis. The aim of this study was to clarify the effects on melatonin synthesis in rats after short term exposure to a 1439 MHz time division multiple access (TDMA) EMF. The average specific absorption ratio (SAR) of the brain was 7.5 W/kg, and the average SARs of the whole body were 1.9 and 2.0 W/kg for male and female rats, respectively. A total of 208 male and female rats were investigated. After acclimatization to a 12 h light-dark (LD) cycle, serum and pineal melatonin levels together with pineal serotonin level under a dark condition (less than 1 lux) were examined by radioimmunoassay. No significant differences in melatonin and serotonin levels were observed between the exposure, sham, and cage control groups. These results suggest that short term exposure to a 1439 MHz TDMA EMF, which is about four times stronger than that emitted by mobile phones, does not alter melatonin and serotonin synthesis in rats. Further investigations on the effects of long term exposure are warranted.     

Individual radiation exposure dose due to support activities at safe shelters in Fukushima Prefecture

Immediately after the accidents in the nuclear power stations in Fukushima on March 11, the Japanese Government ordered the evacuation of the residents within a 20-km radius from the station on March 12, and asked various institutions to monitor the contamination levels of the residents. Hirosaki University, which is located 355 km north of Fukushima City, decided to send support staff to Fukushima. This report summarizes the results of the exposure of 13 individual teams from March 15 to June 20. The support teams surveyed more than 5,000 people during this period. Almost all subjects had external contamination levels of less than 13 kcpm on Geiger-Müller (GM) survey meter, which is categorized as "no contamination level." The 1(st) team showed the highest external exposure dose, but the 4(th) team onward showed no significant change. Subsequently, the internal radiation exposure was measured using a whole body counter that indicated undetectable levels in all staff members. Although the measured external radiation exposure dose cannot have serious biological effects on the health of an individual, a follow-up study of the residents in Fukushima and other regions where the radioactive material has spread will be required for a long time.     

Diversity of RuBisCO gene responsible for CO2 fixation in an Antarctic moss pillar

Antarctic “moss pillars” are lake-bottom biocenoses that are primarily comprised of aquatic mosses. The pillars consist of distinct redox-affected sections: oxidative exteriors and reductive interiors. Batteries of SSU rRNA genotypes of eukaryotes, eubacteria, and cyanobacteria, but no archaea, have been identified in these pillars. However, rRNA-based phylogenetic analysis provides limited information on metabolic capabilities. To investigate the microorganisms that have the potential for CO₂ fixation in the pillars, we studied the genetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, EC 4.1.1.39)—an enzyme involved in CO₂ fixation. PCR clone libraries targeting all forms of the RuBisCO large subunit-encoding gene were constructed and 1,092 clones were randomly sequenced. Phylogenetic analysis indicated that proteobacterial form IA operational RuBisCO units (ORUs) were detected at the same frequency as the cyanobacterial form IB ORUs. Surprisingly, the form IA ORU, which was closely related to the sequences from deep-sea environments, was detected from all moss pillar sections. The form IB ORU related to Bryophyta, considered to be derived from moss, was identical to the sequence of Leptobryum sp. isolated from Lake Hotoke-Ike where the pillars were found. Moreover, certain cyanobacterial ORUs were found exclusively in the exterior of the pillar, whereas form II ORUs related to chemolithoautotrophic sulfur oxidizers and purple sulfur bacteria were found exclusively in the interior. No forms IC, ID, or III RuBisCO genes were detected. This is the first report demonstrating that bacteria with the potential for CO₂ fixation and chemoautotrophy are present in the Antarctic moss pillar ecosystem.     

TRANSPLACENTAL EFFECT OF ETHINYL ESTRADIOL ON MOUSE VAGINAL EPITHELIUM *

The effect of prenatal administration of ethinyl estradiol (EE) on the vaginal epithelium of adult mice was examined histologically. The mice were the offspring of JCL/ICR strain mice given orally 0.02 mg/kg body weight/day or 0.01 mg/kg/day of EE dissolved in olive oil from day 11 to day 17 of gestation at a stage when the urogenital sinus has just appeared in the embryos. The control mice were offspring of those fed with the vehicle alone. Autopsies were performed at 10 to 14 weeks of age. Another group of mice exposed to 0.02 mg/kg/day of EE or vehicle alone in utero, were spayed at 16 weeks of age and killed at 32 weeks of age. In the experimental nonspayed mice, hyperplasia of the vaginal epithelium with intense cornification was seen. The epithelium was significantly thicker than in the controls and showed an EE dose-response relation. One of the 16 mice exposed to 0.01 mg/kg/day of EE in utero had cystic or gland-like structures in the stroma and mucus-secreting cells in the surface epithelium consisting of columnar cells. In some experimental spayed mice, vaginal hyperplasia with cornified epithelium and hypertrophy of the ovarian interstitial tissue without corpus luteum were seen. These results indicate that EE can cross fetal membranes and affect undifferentiated cells in the urogenital sinus and/or Müllerian epithelium.