In vivo neuroprotective role of NMDA receptors against kainate-induced excitotoxicity in murine hippocampal pyramidal neurons

Activation of NMDA receptors has been shown to induce either neuronal cell death or neuroprotection against excitotoxicity in cultured cerebellar granule neurons in vitro. We have investigated the effects of pretreatment with NMDA on kainate-induced neuronal cell death in mouse hippocampus in vivo. The systemic administration of kainate (30 mg/kg), but not NMDA (100 mg/kg), induced severe damage in pyramidal neurons of the hippocampal CA1 and CA3 subfields 3-7 days later, without affecting granule neurons in the dentate gyrus. An immunohistochemical study using an anti-single-stranded DNA antibody and TdT-mediated dUTP nick end labeling analysis both revealed that kainate, but not NMDA, induced DNA fragmentation in the CA1 and CA3 pyramidal neurons 1-3 days after administration. Kainate-induced neuronal loss was completely prevented by the systemic administration of NMDA (100 mg/kg) 1 h to 1 day previously. No pyramidal neuron was seen with fragmented DNA in the hippocampus of animals injected with kainate 1 day after NMDA treatment. The neuroprotection mediated by NMDA was prevented by the non-competitive NMDA receptor antagonist MK-801. Taken together these results indicate that in vivo activation of NMDA receptors is capable of protecting against kainate-induced neuronal damage through blockade of DNA fragmentation in murine hippocampus.     

Physical effects of negative air ions in a wet sauna

 The physical effects of negative air ions on humans were determined in an experimental sauna room equipped with an ionizer. Thirteen healthy persons took a wet sauna bath (dry bulb temperature 42° C, relative humidity 100%, 10 min exposure) with or without negative air ions. The subjects were not told when they were being exposed to negative air ions. There were no differences in the moods of these persons or changes in their blood pressures between the two saunas. The surface temperatures of the foreheads, hands, and legs in the sauna with negative ions were significantly higher than those in the sauna without ions. The pulse rates and sweat produced in the sauna with ions were singificantly higher than those in the sauna without ions. The results suggest that negative ions may amplify the effects on humans of the sauna. Received: 31 March 1995 / Revised: 25 July 1995 / Accepted: 26 July 1996     

Molecular cloning of Chinese hamster ceramide glucosyltransferase and its enhanced expression in peroxisome-defective mutant Z65 cells

To clarify the metabolic bases of characteristic increases in the concentrations of glucosylceramide (CMH) and GM3 in peroxisome-defective mutant Chinese hamster ovary (CHO) cells (Z65), we measured the ceramide glucosyltransferase (CGT) and beta-glucosidase activities in Z65 and CHO-K1 cells, and found that the former enzyme was responsible for the accumulation of CMH in Z65 cells. Inhibition of CGT by D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) caused a marked reduction in a incorporation of [3-14C]serine to CMH in both CHO-K1 and Z65 cells, but resulted in the accumulation of ceramide in Z65 cells in a concentration higher than that in CHO-K1 cells. Then, we cloned the cDNA encoding CGT from CHO-K1 cells, which exhibited sequence homology with the human gene product (98.7%). Northern blot analysis of CGT revealed increased expression of it in Z65 cells compared with that in CHO-K1 cells, which probably caused the simultaneous increase in GM3. With an immunohistochemical procedure, GM3 was found to be more strongly expressed in the cell membrane of Z65 cells than in CHO-K1 cells.     

DNA augments antigenicity of mycobacterial DNA-binding protein 1 and confers protection against Mycobacterium tuberculosis infection in mice

Mycobacterium consists up to 7% of mycobacterial DNA-binding protein 1 (MDP1) in total cellular proteins. Host immune responses to MDP1 were studied in mice to explore the antigenic properties of this protein. Anti-MDP1 IgG was produced after infection with either bacillus Calmette-Guérin or Mycobacterium tuberculosis in C3H/HeJ mice. However, the level of Ab was remarkably low when purified MDP1 was injected. MDP1 is considered to be associated with DNA in nucleoid, which contains immunostimulatory CpG motif. Therefore, we examined coadministration of MDP1 and DNA derived from M. tuberculosis. Consequently, this procedure significantly enhanced the production of MDP1-specific IgG. Five nanograms of DNA was enough to enhance MDP1-specific IgG production in the administration of 5 microg of MDP1 into mice. Strong immune stimulation by such a small amount of DNA is noteworthy, because >1,000- to 100,000-fold doses of CpG DNAs are used for immune activation. A synthetic peptide-based study showed that B cell epitopes were different between mice administered MDP1 alone and those given a mixture of MDP1 and DNA, suggesting that DNA alters the three-dimensional structure of MDP1. Coadministration of DNA also enhanced MDP1-specific IFN-gamma production and reduced the bacterial burden of a following challenge of M. tuberculosis, showing that MDP1 is a novel vaccine target. Finally, we found that MDP1 remarkably enhanced TLR9-dependent immune stimulation by unmethylated CpG oligo DNA in vitro. To our knowledge, MDP1 is the first protein discovered that remarkably augments the CpG-mediated immune response and is a potential adjuvant for CpG DNA-based immune therapies.     

Relative efficacies of 1,4-diazepines on GABA-stimulated chloride influx in rat brain vesicles

The effects of 1,4-diazepines with two annelated heterocycles [brotizolam (WE 941), ciclotizolam (WE 973) and WE 1008] on gamma-aminobutyric acid (GABA)-stimulated chloride influx into rat brain membrane vesicles were examined. Brotizolam enhanced GABA (30 microM)-stimulated 36Cl- influx (146.1% of control), while ciclotizolam and WE 1008 showed only a small enhancement (119.3% and 119.1%, respectively) of GABA-stimulated 36Cl- uptake. Brotizolam resulted in a left shift of the GABA dose response curve at lower concentrations of GABA (10 microM), while at higher concentrations of GABA (1 mM), brotizolam caused a reduction of the maximal response. The enhancement of GABA-stimulated 36Cl- uptake by brotizolam (0.1 microM) was antagonized by Ro 15-1788. At higher concentration of GABA (300 microM), brotizolam inhibited GABA-stimulated 36Cl- uptake in a dose dependent manner and Ro15-1788 failed to antagonize this effect. These results suggest that 1) brotizolam produces an enhancement of GABA (30 microM)-stimulated chloride influx through the benzodiazepine receptor. 2) brotizolam inhibition of GABA (300 microM)-stimulated chloride influx involves an additional mechanism, and 3) the sedative-hypnotic action of brotizolam may be related to its high efficacy at the benzodiazepine/GABA-gated chloride channel.     

Redesign of coenzyme B(12) dependent diol dehydratase to be resistant to the mechanism-based inactivation by glycerol and act on longer chain 1,2-diols

Coenzyme B(12) dependent diol dehydratase undergoes mechanism-based inactivation by glycerol, accompanying the irreversible cleavage of the coenzyme Co-C bond. Bachovchin et al. [Biochemistry16, 1082-1092 (1977)] reported that glycerol bound in the G(S) conformation, in which the pro-S-CH(2) OH group is oriented to the hydrogen-abstracting site, primarily contributes to the inactivation reaction. To understand the mechanism of inactivation by glycerol, we analyzed the X-ray structure of diol dehydratase complexed with cyanocobalamin and glycerol. Glycerol is bound to the active site preferentially in the same conformation as that of (S)-1,2-propanediol, i.e. in the G(S) conformation, with its 3-OH group hydrogen bonded to Serα301, but not to nearby Glnα336. k(inact) of the Sα301A, Qα336A and Sα301A/Qα336A mutants with glycerol was much smaller than that of the wild-type enzyme. k(cat) /k(inact) showed that the Sα301A and Qα336A mutants are substantially more resistant to glycerol inactivation than the wild-type enzyme, suggesting that Serα301 and Glnα336 are directly or indirectly involved in the inactivation. The degree of preference for (S)-1,2-propanediol decreased on these mutations. The substrate activities towards longer chain 1,2-diols significantly increased on the Sα301A/Qα336A double mutation, probably because these amino acid substitutions yield more space for accommodating a longer alkyl group on C3 of 1,2-diols. Database Structural data are available in the Protein Data Bank under the accession number 3AUJ. Structured digital abstract ? Diol dehydrase gamma subunit, Diol dehydrase beta subunit and Diol dehydrase alpha subunit physically interact by X-ray crystallography (View interaction).     

Effect of 24,25-dihydroxyvitamin D3 on 1,25-dihydroxyvitamin D3 metabolism in calcium-deficient rats.

The effect of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] metabolism was examined in rats fed on a low-calcium diet. These rats exhibit hypocalcaemia, high urinary cyclic AMP excretion, a markedly elevated serum 1,25(OH)2D concentration and low serum concentrations of both 24,25(OH)2D and 25(OH)D. When the rats are treated orally with 1, 5 or 10 micrograms of 24,25(OH)2D3/100 g every day, there is a dramatic decrease in serum 1,25(OH)2D concentration in a dose-dependent manner concomitant with an increase in serum 24,25(OH)2D concentration. Serum calcium concentration and urinary cyclic AMP excretion are not significantly affected by the 24,25(OH)2D3 treatment, which suggests that parathyroid function is not affected by the 24,25(OH)2D3 treatment. The 25(OH)D3 1 alpha-hydroxylase activity measured in kidney homogenates is markedly elevated in rats on a low-calcium diet but is not affected by any doses of 24,25(OH)2D3. In contrast, recovery of intravenously injected [3H]1,25(OH)2D3 in the serum is decreased in 24,25(OH)2D3-treated rats. Furthermore, when [3H]1,25(OH)2D3 is incubated in vitro with kidney or intestinal homogenates of 24,25(OH)2D3-treated rats there is a decrease in the recovery of radioactivity in the total lipid extract as well as in the 1,25(OH)2D3 fraction along with an increase in the recovery of radioactivity in the water-soluble phase. These results are consistent with the possibility that 24,25(OH)2D3 has an effect on 1,25(OH)2D3 metabolism, namely that of enhancing the degradation of 1,25(OH)2D3. However, because a considerable proportion of the injected 24,25(OH)2D3 is expected to be converted into 1,24,25(OH)3D3 by renal 1 alpha-hydroxylase in 24,25(OH)2D3-treated rats, at least a part of the decrease in serum 1,25(OH)2D concentration may be due to a competitive inhibition by 24,25(OH)2D3 of the synthesis of 1,25(OH)2D3 from 25(OH)D3. Thus the physiological importance of the role of 24,25(OH)2D3 in regulating the serum 1,25(OH)2D concentration as well as the mechanism and metabolic pathway of degradation of 1,25(OH)2D3 remain to be clarified.     

Absorption of mulberry root urease to the hemolymph of the silkworm, Bombyx mori

Mulberry leaves are the sole diet of the silkworm, Bombyx mori. The host urease is incorporated into the larval hemolymph and involved in nitrogen metabolism in the insect. To investigate the selective absorption of the host urease to the larvae, crude urease was prepared from mulberry leaves and roots. Root urease was identical to leaf urease on the basis of electrophoretic analyses: (1) the urease activity appeared in the same migration position in a native gel; (2) There was no difference in molecular mass of the subunit. The root urease was orally injected to the fifth instar larvae of the silkworm. Just before spinning, the larvae absorbed intact urease from the midgut lumen to the hemolymph without the loss of activity. The capacity to absorb urease occurred only at the specific stage. Localization of host urease in midgut tissue was observed using confocal laser scanning microscopy and transmission electron microscopy. Based on spatial distribution of immunofluorescent signals and immunogold particles, host urease specifically attached to the surfaces of microvilli existing in the apical side of columnar cells and appeared in the cytoplasm of the cells for transport to the hemolymph. The incorporation efficiency of root urease into the hemolymph was significantly higher than for ureases from jack bean seeds and Bacillus pasteurii. The urease that was transported to the hemolymph was electrophoretically altered, compared with the host urease extracted.     

Serum and tissue coenzyme Q9 in rats with thyroid dysfunctions

Serum and tissue CoQ9 levels were determined in hypothyroid, euthyroid and hyperthyroid rats. A significant negative correlation was demonstrated between serum FT4 or T3 and CoQ9 in rats with various states of thyroid functions. Liver CoQ9 was significantly increased in rats rendered mildly hyperthyroid. There was a significant positive correlation between serum FT4 or T3 and liver CoQ9. While liver CoQ9 did not significantly change in severely hyperthyroid animals, liver mitochondrial CoQ9 showed a significant positive correlation with serum T3. Kidney and heart CoQ9 levels did not significantly change in hyperthyroid rats, but those in hypothyroid rats showed a tendency to increase. It was suggested that the synthesis of CoQ9 was increased in the liver in hyperthyroidism.     

In Vitro Culture of a Root Parasite, Aeginetia indica L. II. The Plane of Cell Division in the Tendril

Factors affecting septation (cell division) of the tendril whichfacilitates the organic connection with the host were studiedin a root parasite Aeginetia indica L. Transverse cell division,which occurs perpendicular to the long axis of the tendril,was promoted by additions of sucrose, glucose and cytokininsto the basal medium. Longitudinal cell division of the tendril,which takes place parallel or obliquely to the long axis, wasstimulated by cytokinins, but not by sucrose. The latter typeof cell division was frequent in basal and sub-basal cells ofthe tendril but was extremely rare in apical cells. The orientationof the planes of these cell divisions was closely related tocell shape. Abnormal growth of the tendril was seen in germinatingseeds grown for six weeks or more in media containing both Miscanthus(a host) root extract and cytokinin. (Received February 23, 1984; Accepted June 12, 1984)