Neuroprotective effects of yokukansan, a traditional Japanese medicine, on glutamate-mediated excitotoxicity in cultured cells.

To clarify the mechanism of yokukansan (TJ-54), a traditional Japanese medicine, against glutamate-mediated excitotoxicity, the effects of TJ-54 on glutamate uptake function were first examined using cultured rat cortical astrocytes. Under thiamine-deficient conditions, the uptake of glutamate into astrocytes, and the levels of proteins and mRNA expressions of glutamate aspartate transporter of astrocytes significantly decreased. These decreases were ameliorated in a dose-dependent manner by treatment with TJ-54 (100-700 microg/ml). The improvement of glutamate uptake with TJ-54 was completely blocked by the glutamate transporter inhibitor DL-threo-beta-hydroxyaspartic acid. Effects of TJ-54 on glutamate-induced neuronal death were next examined by using cultured PC12 cells as a model for neurons. Addition of 17.5 mM glutamate to the culture medium induced an approximately 50% cell death, as evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. TJ-54 (1-1000 microg/ml) inhibited the cell death in a dose-dependent manner. Furthermore, competitive binding assays to glutamate receptors showed that TJ-54 bound potently to N-methyl-D-aspartate receptors, in particular, to its glutamate and glycine recognition sites. These results suggest that TJ-54 may exert a neuroprotective effect against glutamate-induced excitotoxicity not only by amelioration of dysfunction of astrocytes but also by direct protection of neuronal cells.

Motor pattern of the stinging response in the honeybee Apis mellifera

In the stinging response of the worker honeybee (Apis mellifera), rhythmic movements of the lancets on the stylet are produced by alternating contractions of a set of stinging muscles (a protractor, M198, and a retractor, M199) on each side during co-contraction of the frucula muscles (M197s) on both sides. In this study, stinging movements were elicited by tactile stimulation to the sternum in isolated abdomens, in intact animals and in preparations in which the connectives between the sixth and terminal abdominal ganglia were cut. There was a close relationship among the following three temporal variables of stinging motoneurone pattern: the interval between successive bursts of a stinging muscle, the duration of a burst and the time lag between the bursts of homologous stinging muscles on both sides. All of these variables increased linearly as the sting was inserted deeper into a soft object and the tension on the lancets increased. When sensory nerves from the proprioceptors (campaniform sensilla on the tapering sting shaft and hair plates at the basal cuticular plate) were cut on both sides, the relative timing of bursts of homologous stinging muscles on both sides and antagonistic stinging muscles on each side became more variable. When a proprioceptive input was removed from one side during penetration of the sting, the frequency of the bursts of stinging muscles was higher and the duration of bursts was shorter on the cut side than on the intact side; nevertheless, a sting muscle was still activated out of phase with its antagonistic muscle on the ipsilateral side and its homologous muscle on the contralateral side. These results suggest that the motor pattern driving the rhythmic movements of stinging muscles is produced by a central pattern generator consisting of a pair of oscillators located in the terminal abdominal ganglion and that the precise timing of the motor pattern in a hemiganglion is controlled mainly by proprioceptive inputs on its own side.

Two cis-regulatory elements that mediate different signaling pathways for serum-dependent activation of the junB gene.

Transcription of the junB gene is rapidly and transiently induced by a variety of extracellular signals. We report here that expression directed by a junB promoter/chloramphenicol acetyltransferase reporter construct (junB/CAT) is induced by fetal bovine serum, 12-O-tetradecanoylphorbol-13-acetate (TPA), epidermal growth factor (EGF), platelet-derived growth factor, and fibroblast growth factor in mouse fibroblast 3T6 cells. Deletion analysis of the promoter region of the junB gene indicates that there are at least two cis-regulatory elements that confer the capacity for serum-dependent induction. These two serum response elements (SRE1 and SRE2) are mapped between nucleotides -1451 and -1425 and between nucleotides -3100 and -2500, respectively, relative to the site of initiation of transcription. SRE1, the nucleotide sequence of which resembles that of the serum response element of the c-fos gene, is activated by TPA, platelet-derived growth factor, and fibroblast growth factor, but these growth-stimulating factors do not induce SRE2-mediated transcription. Pretreatment of the cells with phorbol dibutyrate, which reduces the level of protein kinase C activity in cells, almost completely abolishes the activation of SRE1 by TPA. Pretreatment with phorbol dibutyrate also reduces (but does not eliminate) the serum-dependent activation of SRE1. By contrast, the induction of SRE2 by serum is not affected by this pretreatment. Herbimycin A, an inhibitor of protein kinases, inhibits the activity of SRE2, but not that of SRE1. These results suggest that transcription of the junB gene can be induced by at least two distinct signaling pathways, which are mediated by SRE1 and SRE2, respectively. In addition, EGF induces expression of junB/CAT as strongly as does serum, but neither SRE1 nor SRE2 is sufficient for responsiveness to EGF.

Transcriptional regulation of the c-jun gene by retinoic acid and E1A during differentiation of F9 cells.

Differentiation of mouse F9 embryonal carcinoma (EC) cells can be induced by exposure to retinoic acid (RA) or by expression of adenovirus E1A. The transcription of the c-jun gene is stimulated by either RA or E1A. We report here that both RA and E1A strongly induce the expression of chloramphenicol acetyltransferase (CAT) from c-jun promoter/CAT reporter construct (c-jun/CAT), which is stably integrated into F9 cells, in a manner that is independent of both copy number and integration locus. The induction of c-jun/CAT expression is observed in undifferentiated F9 cells, but not in differentiated F9 cells, adenovirus-infected F9 cells or HeLa cells. Deletion analysis of the promoter region of the c-jun gene indicates that the sequence elements required for the RA- and E1A-mediated induction are identical and they have been defined as a region of 145 bp between -190 and -46 of the 5' flanking region of c-jun. This RA and E1A response element (RERE) contains five variants of the motif CGCGGTGACGNT. The upstream two motifs are adjacent and extend in opposite directions, creating an imperfect palindrome. The downstream four motifs are located at 35 or 36 bp intervals in the same orientation. Substitution and insertion analysis indicates that these motifs and their regular intervals are important for the activity of the RERE.

Deficiency of glycosyl-phosphatidylinositol anchored proteins on paroxysmal nocturnal haemoglobinuria (PNH) neutrophils and monocytes: heterogeneous deficiency of decay-accelerating factor (DAF) and CD16 on PNH neutrophils.

Glycosyl-phosphatidylinositol (GPI) anchored membrane proteins have been reported to be deficient on affected paroxysmal nocturnal haemoglobinuria (PNH) blood cells. In the present study we investigated the deficiency of several GPI anchored membrane proteins on PNH neutrophils (PMN) and monocytes from 10 patients with PNH. Decay-accelerating factor (DAF) and Fc gamma R-III (CD16) on PMN, DAF and CD14 on monocytes, were investigated by two-colour immunofluorocytometry. Neutrophil alkaline phosphatase activity was also assayed on PNH neutrophils. Normal human PMN were always shown phenotypically to be DAF+/CD16+. A DAF-/CD16- subpopulation of PMN was demonstrated in all the patients studied. In six out of the 10 patients, deficiencies of DAF and CD16 were found simultaneously on affected PNH PMN. The percentage of DAF- PMN showed a positive correlation with the neutrophil alkaline phosphatase (NAP) score. However, it should be noted that, in four out of the 10 patients with PNH, a DAF+/CD16- subpopulation of PMN was also clearly found. This may indicate that the deficiencies of DAF and CD16 on PNH PMN are heterogeneous. Normal human monocytes were demonstrated to be DAF+/CD14+, whereas PNH monocytes consisted of subpopulations of DAF+/CD14+ and DAF-/CD14-. In the same patients with PNH, the deficiencies of DAF on PMN and monocytes correlated well with each other. These results suggest that, at least in some patients with PNH, the mechanisms which induce the membrane defects of PNH blood cells are heterogeneous.

Decreased frequency of bone marrow NK progenitors in aplastic anaemia.

Twelve patients with aplastic anaemia were studied with regard to the frequency of NK progenitors in the bone marrow (BM) to investigate the mechanism of depressed NK activity and low NK cell count in the peripheral blood. NK cell (CD16+ cell) count and NK (K562) activity were significantly decreased (P less than 0.02 and P less than 0.001, respectively) in the patients as compared to eight healthy control subjects. Nylon wool non-adherent (NW-NA) BM mononuclear cells (MNC) of each patient and control were prepared. Mature T and NK cells were extensively depleted by sheep red cell rosette formation followed by a centrifugation on Ficoll-Hypaque and monoclonal antibody mediated complement dependent cytolysis. Those selected BM cells were cultured in the presence of recombinant interleukin 2 (rIL2). Generation of NK activity was significantly decreased (P less than 0.01) in the patients with aplastic anaemia. Frequency of NK progenitors in the selected BM cells assayed by the limiting dilution method was significantly decreased in those patients (P less than 0.05). The frequency of BM NK progenitors relative to NW-NA BM cells were related to NK cell count (P less than 0.01). Those results indicate that depressed NK activity in aplastic anaemia is closely related to decreased NK cell count which is probably due to decreased production of NK cells in the BM.

Characterization of natural killer cells cultured from human bone marrow cells.

Human bone marrow (BM) cells, depleted of nylon wool-adherent cells, T cells, and natural killer (NK) cells, were cultured in medium containing recombinant interleukin 2 (rIL2). After 21 or 24 days in culture, numerous lymphoid cells with multiple azurophilic granules and a morphology similar to large granular lymphocytes (LGL) were found. Two-color analysis of surface phenotype showed many of these cells to be NKH1-positive and a limited number of cells had other NK markers such as CD16, CD2, or CD8. The CD3 antigen was not coexpressed with NKH1. The cultured BM cells were cytotoxic for K562, Daudi, and Raji cell lines. The NKH1+, CD2-, CD3-, CD16- cells were sorted and, in addition to having the LGL morphology, were found to be cytotoxic for K562 cells (NK [K562]). The generation of NK(K562) activity was significantly suppressed by 5-bromodeoxyuridine plus ultraviolet light treatment, indicating that DNA synthesis is required. These experiments suggest that the described culture conditions allow differentiation of progenitor cells, into immature, but functionally active, NK cells.

Prevention of doxorubicin myocardial toxicity in mice by reduced glutathione.

The effect of reduced glutathione (GSH) on acute myocardial toxicity due to doxorubicin (DXR) was investigated. At 20 mg/kg i.p. DXR was 100% lethal to BALB/c X DBA/2 F1 mice. At 500 or 1000 mg/kg i.p. GSH significantly decreased the lethality (P less than 0.01). Electron micrographs of the myocardium from DXR-treated mice showed narrowing of myofibrils, edematous cytoplasm, and mitochondrial swelling which were detectable at day 2, was strongest at day 14, but had disappeared by day 56. Light microscopic examination revealed that intercellular spaces between myocardial cells were widened at day 56, indicating shrinking of myocardial cells. These changes were significantly decreased by treatment with GSH (500 mg/kg i.p.). Treatment with DXR (14 mg/kg) significantly decreased myocardial non-protein sulfhydryl content (P less than 0.02) and administration of GSH (500 mg/kg) prevented the drop of non-protein sulfhydryl levels due to DXR treatment. Thus it was considered that administration of exogenous GSH contributes to prevention of the acute myocardial toxicity of DXR by increasing extracellular GSH levels and intracellular GSH synthesis, which detoxifies DXR-oxygen metabolites. The administration of GSH did not interfere with the antineoplastic effect of DXR against L1210 mouse leukemia.