Effects of slightly acidic electrolysed drinking water on mice

Slightly acidic electrolysed (SAE) water is a sanitizer with strong bactericidal activity due to hypochlorous acid. We assessed the safety of SAE water as drinking water for mice at a 5 ppm total residual chlorine (TRC) concentration to examine the possibility of SAE water as a labour- and energy-saving alternative to sterile water. We provided SAE water or sterile water to mice for 12 weeks, during which time we recorded changes in body weight and weekly water and food intakes. At the end of the experiment, all of the subject animals were sacrificed to assess serum aspartate aminotransferase, alanine aminotransferase and creatinine levels and to examine the main organs histopathologically under a light microscope. In addition, we investigated the bacteria levels of both types of water. We found no difference in functional and morphological health condition indices between the groups. Compared with sterile water, SAE water had a relatively higher ability to suppress bacterial growth. We suggest that SAE water at 5 ppm TRC is a safe and useful alternative to sterile water for use as drinking water in laboratory animal facilities.     

A novel action of collapsin: Collapsin-1 increases antero- and retrograde axoplasmic transport independently of growth cone collapse

Chick collapsin-1, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Collapsin-1 induces growth cone collapse via a pathway which may include CRMP-62 and heterotrimeric G proteins. CRMP-62 protein is related to UNC-33, a nematode neuronal protein required for appropriately directed axonal extension. Mutations in unc-33 affect neural microtubules, the basic cytoskeletal elements for axoplasmic transport. Using computer-assisted video-enhanced differential interference contrast microscopy, we now demonstrate that collapsin-1 potently promotes axoplasmic transport. Collapsin-1 doubles the number of antero- and retrograde-transported organelles but not their velocity. Collapsin-1 decreases the number of stationary organelles, suggesting that the fraction of time during which a particle is moving is increased. Collapsin-1-stimulated transport occurs by a mechanism distinct from that causing growth cone collapse. Pertussis toxin (PTX) but not its B oligomer blocks collapsin-induced growth cone collapse. The holotoxin does not affect collapsin-stimulated axoplasmic transport. Mastoparan and a myelin protein NI-35 induce PTX-sensitive growth cone collapse but do not stimulate axoplasmic transport. These results provide evidence that collapsin has a unique property to activate axonal vesicular transport systems. There are at least two distinct pathways through which collapsin exerts its actions in developing neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 316–328, 1997     

Identification of the three-dimensional pharmacophore of κ-opioid receptor agonists

A selective κ-opioid receptor agonist might act as a powerful analgesic without the side effects of μ-opioid receptor-selective drugs such as morphine. The eight classes of known κ-opioid receptor agonists have different chemical structures, making it difficult to construct a pharmacophore model that takes them all into account. Here we propose a new three-dimensional pharmacophore model that encompasses the κ-activities of all classes, which utilizes conformational sampling of agonists by high-temperature molecular dynamics and pharmacophore extraction through a series of molecular superpositions.     

Gait Variability before Surgery and at Discharge in Patients Who Undergo Total Knee Arthroplasty: A Cohort Study

This study aimed to determine gait ability at hospital discharge in patients undergoing total knee arthroplasty (TKA) as an indicator of the risk of falling. Fifty-seven patients undergoing primary TKA for knee osteoarthritis participated in this study. Gait variability measured with accelerometers and physical function including knee range of motion (ROM), quadriceps strength, walking speed, and the Timed Up and Go (TUG) test were evaluated preoperatively and at discharge from the hospital (1 month before and 5 days after surgery). All patients were discharged directly home at 5 days after surgery. Knee flexion of ROM, quadriceps strength, walking speed, and the TUG test results were significantly worse at hospital discharge than preoperatively (p < 0.001). However, gait variability was not significantly different before and after TKA. This result indicated that patients following TKA surgery could walk at hospital discharge as stably as preoperatively regardless of the decrease in physical function, including knee ROM, quadriceps strength, and gait speed after surgery.     

Orally tolerant CD4 T cells respond poorly to antigenic stimulation but strongly to direct stimulation of intracellular signaling pathways

The response of splenic CD4 T cells from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic mice after long-term feeding of a diet containing this antigen was examined. These CD4 T cells exhibited a decreased response to OVA peptide stimulation, in terms of proliferation, interleukin-2 secretion, and CD40 ligand expression, compared to those from mice fed a control diet lacking OVA, demonstrating that oral tolerance of T cells had been induced through oral intake of the antigen. We investigated the intracellular signaling pathways, which were Ca/CN cascade and Ras/MAPK cascade, of these tolerant CD4 T cells using phorbol-12-myristate-13-acetate (PMA) and ionomycin, which are known to directly stimulate these pathways. In contrast to the decreased response to TCR stimulation by OVA peptide, it was shown that the response of splenic CD4 T cells to these reagents in the state of oral tolerance was stronger. These results suggest that splenic CD4 T cells in the state of oral tolerance have an impairment in signaling, in which signals are not transmitted from the TCR to downstream signaling pathways, and have impairments in the vicinity of TCR. This revised version was published online in July 2006 with corrections to the Cover Date.     

Absence of Nceh1 augments 25-hydroxycholesterol-induced ER stress and apoptosis in macrophages

An excess of cholesterol and/or oxysterols induces apoptosis in macrophages, contributing to the development of advanced atherosclerotic lesions. In foam cells, these sterols are stored in esterified forms, which are hydrolyzed by two enzymes: neutral cholesterol ester hydrolase 1 (Nceh1) and hormone-sensitive lipase (Lipe). A deficiency in either enzyme leads to accelerated growth of atherosclerotic lesions in mice. However, it is poorly understood how the esterification and hydrolysis of sterols are linked to apoptosis. Remarkably, Nceh1-deficient thioglycollate-elicited peritoneal macrophages (TGEMs), but not Lipe-deficient TGEMs, were more susceptible to apoptosis induced by oxysterols, particularly 25-hydroxycholesterol (25-HC), and incubation with 25-HC caused massive accumulation of 25-HC ester in the endoplasmic reticulum (ER) due to its defective hydrolysis, thereby activating ER stress signaling such as induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). These changes were nearly reversed by inhibition of ACAT1. In conclusion, deficiency of Nceh1 augments 25-HC-induced ER stress and subsequent apoptosis in TGEMs. In addition to reducing the cholesteryl ester content of foam cells, Nceh1 may protect against the pro-apoptotic effect of oxysterols and modulate the development of atherosclerosis.     

Nitric Oxide Participates in the Stimulatory and Neurotoxic Action of Endothelin on Rat Striatal Dopaminergic Neurons

1. Our method of real-time monitoring of dopamine release from rat striatal slices revealed that endothelin (ET)-3-induced dopamine release was inhibited by N ^G-methyl-L-arginine (L-NMMA; 1 mM), an inhibitor of nitric oxide (NO) synthase, while N ^G-methyl-D-arginine (D-NMMA; 1 mM), an inactive isomer of L-NMMA, had no effect.2. The inhibition of L-NMMA (0.1 mM) became apparent when tissues were pretreated with tetrodotoxin (1 M) for 30 min and subsequently exposed to ET-3 (4 M).3. L-NMMA (0.1 and 1 mM) dose dependently protected against ET-3-triggered hypoxic/hypoglycemic impairment of striatal responses to high K⁺.4. Thus, NO may work as a promoter in mediation of the stimulatory and neurotoxic action of ET-3 on the striatal dopaminergic system, presumably by interacting with interneurons in the striatum.     

Enhancement of penicillin acylase activity by cultivating immobilized Kluyvera citrophila

Summary Whole cells of Kluyvera citrophila were immobilized in polyacrylamide gel. The penicillin acylase activity of immobilized whole cells was 60%–70% of native cells. When the immobilized cells were continuously cultivated for 40 h in an aerated fermentor containing peptone medium and were treated with alkali in order to remove -lactamase activity, the immobilized cells produced ampicillin up to 4.4 times faster than noncultivated cells.Ampicillin production was investigated in a column system using these cultivated immobilized whole cells. The cultivated immobilized cells showed excellent performance in continuous ampicillin production.     

GABAergic inhibition regulates developmental synapse elimination in the cerebellum

Functional neural circuit formation during development involves massive elimination of redundant synapses. In the cerebellum, one-to-one connection from excitatory climbing fiber (CF) to Purkinje cell (PC) is established by elimination of early-formed surplus CFs. This process depends on glutamatergic excitatory inputs, but contribution of GABAergic transmission remains unclear. Here, we demonstrate impaired CF synapse elimination in mouse models with diminished GABAergic transmission by mutation of a single allele for the GABA synthesizing enzyme GAD67, by conditional deletion of GAD67 from PCs and GABAergic interneurons or by pharmacological inhibition of cerebellar GAD activity. The impaired CF synapse elimination was rescued by enhancing GABA(A) receptor sensitivity in the cerebellum by locally applied diazepam. Our electrophysiological and Ca2+ imaging data suggest that GABA(A) receptor-mediated inhibition onto the PC soma from molecular layer interneurons influences CF-induced Ca2+ transients in the soma and regulates CF synapse elimination from postnatal day 10 (P10) to around P16.