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Abstract. We have recently reported that nobiletin, a citrus flavonoid, improves impaired memory in olfactory-bulbectomized (OBX) mice, which have been widely utilized as a useful paradigm that shares some major clinical features of Alzheimer's disease. Here, we examined the effects of nobiletin on OBX-induced cholinergic neurodegeneration in mice. OBX mice showed reduced acetylcholinesterase (AChE) staining and choline acetyltransferase (ChAT) expression in the hippocampus. An 11-day administration of nobiletin rescued OBX-induced decrease in the density of AChE-staining and ChAT expression in the hippocampus. These results suggest that nobiletin rescues OBX-induced cholinergic neurodegeneration, accompanied by improvement of impaired memory in OBX mice.

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In normal non‐exercised skeletal muscles in mice, the activity of histidine decarboxylase (HDC), the enzyme which forms histamine, was very low. HDC activity in the quadriceps femoris muscle was markedly elevated following contractions evoked by even a few minutes of direct electrical stimulation, peaking at 8‐12 h following contraction lasting 10 min, and gradually decreasing during the 24 h following contraction. The elevation in HDC activity depended on the duration and strength of stimulation. Direct electrical stimulation induced a quantitatively similar elevation of HDC activity in the muscles of mast‐cell‐deficient mice (W/Wv mice). Prolonged walking at a speed of 6 m min−1 for up to 6 h with a 30 min rest period at 3 h also elevated muscle HDC activity, the magnitude of the elevation being related to the duration of the walking. Repeated exercise (training) for several days diminished the elevation of muscle HDC activity induced by walking. In contrast, starvation augmented the elevation of muscle HDC activity induced by walking. Intraperitoneal injection of interleukin‐1β (IL‐1β) also elevated muscle HDC activity in a dose‐dependent manner, as little as 1 μg kg−1 of IL‐1 producing a significant elevation of muscle HDC activity. IL‐1β was immunohistochemically detected in normal non‐exercised quadriceps femoris muscle. We could not detect a significant increase in IL‐1β after exercise in the muscle or in serum: it may be below the level of detection. On the basis of these results, together with those reported previously and the known actions of histamine, we propose that an elevation of HDC activity and generation of histamine occur in skeletal muscle following muscle contraction possibly as a result of induction by IL‐1β and that the histamine may be involved in fatigue in skeletal muscle as part of a defence mechanism preventing damage to the muscle.

Olfactory bulbectomized (OBX) mice showed significant impairment of learning and memory-related behaviors 14 days after olfactory bulbectomy, as measured by passive avoidance and Y-maze tasks. We here observed a large impairment of hippocampal long-term potentiation (LTP) in the OBX mice. Concomitant with decreased acetylcholinesterase expression, protein kinase C (PKC)a autophosphorylation and NR1(Ser-896) phosphorylation significantly decreased in the hippocampal CA1 region of OBX mice. Both PKCa and NR1(Ser-896) phosphorylation significantly increased following LTP in the control mice, whereas increases were not observed in OBX mice. Like PKC activities, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in the hippocampal CA1 region of OBX mice as compared with that of control mice. In addition, increased CaMKII autophosphorylation following LTP was not observed in OBX mice. Finally, the impairment of CaMKII autophosphorylation was closely associated with reduced pGluR1(Ser-831) phosphorylation, without change in synapsin I (site 3) phosphorylation in the hippocampal CA1 region of OBX mice. Taken together, in OBX mice NMDA receptor hypofunction, possibly through decreased PKCa activity, underlies decreased CaMKII activity in the post-synaptic regions, thereby impairing LTP induction in the hippocampal CA1 region. Both decreased PKC and CaMKII activities with concomitant LTP impairment account for the learning disability observed in OBX mice. Keywords: calcium/ calmodulin-dependent protein kinase II, long-term potentiation, NMDA, olfactory bulbectomy, protein kinase C.

Propolis is a resinous material gathered by honeybees from the buds and bark of certain trees and plants. In Japan, propolis is widely used as a health food and the Japanese believe that it can cure inflammation, heart disease, and even diabetes and cancer. Biological properties of propolis have become a point of particular interest recently.1) The most used formulation in folk medicine is the ethanol extract. 2)Chemical analysis indicated that propolis is a multicomponent mixture of various compounds with prevalence of flavonoids and phenolic acids.3) Several biological attributes such as anticancer, antioxidant, antimicrobial, anti-inflammatory and antibiotic activities have been reported for propolis.4) The standardization of propolis preparations is indeed difficult because of changes in chemical composition and pharmacological activities, resulting from variation in geographical and botanical origin.5) It is important to investigate their mechanisms of action in order to predict possible therapeutic and toxic effects, and to also use this information to develop and design new drugs that are even more effective for the prevention and treatment of cancer. We are interested in the effects of various natural products on cell growth in human cancer cells, as predictors of novel agents that may be useful in cancer chemoprevention or therapy.6) In this study, the inhibitory effects of propolis, a new preparation (CB Propolis) isolated from Brazilian propolis, on the growth of the human leukemia cell line U937 and on the synthesis of DNA, RNA and protein in U937 cells are discussed. MATERIALS AND METHODSChemicals Aqueous solution of propolis, 7) CB Propolis (the dried ethanol extract of Brazilian propolis), was obtained from a commercial supplier (ChatBlanc inc., Tokyo, Japan). Lyophilized CB Propolis is equivalent to 18.5 mg propolis per 100 ml. In this study propolis was diluted in dimethysulfoxide (DMSO) and filtered with a sterile filter prior to use, and then added at the appropriate final concentrations to cultures of U937 cells. Control cells were treated with the same amount of vehicle alone. The final DMSO concentrations never exceeded 0.5% (v/v). In this condition, we confirmed that apoptosis was not observed in U937 cells. Z-Asp-CH 2 -DCB (a wide-spectrum caspase inhibitor) was purchased from Peptide Institute (Osaka, Japan). The cells were treated with 100 mM inhibitor for 1 h before the treatment with propolis. [ Cell Lines and Cell Culture To examine the effects of propolis on cell proliferation, U937 human histiocytic lymphoma cells were obtained from the Japanese Research Resources Bank, Tokyo, Japan. The cells (4ϫ10 6 cells/ml) were grown in RPMI 1640 medium (Iwaki Co., Ltd, Tokyo) supplemented with 100 units/ml of penicillin, 100 mg/ml streptomycin and 10% heat-inactivated fetal bovine serum at 37°C under a humidified 95% air 5% CO 2 atmosphere, and passaged every 7 d. U937 cells had a doubling time of about 24-30 h under these conditions.Cell Proliferation Experiments Cells were inoculated at a d...

BackgroundIt has been demonstrated that angiotensin II (Ang II) participates in either the inhibition or the facilitation of nociceptive transmission depending on the brain area. Neuronal Ang II is locally synthesized not only in the brain, but also in the spinal cord. Though the spinal cord is an important area for the modulation of nociception, the role of spinal Ang II in nociceptive transmission remains unclear. Therefore, in order to elucidate the role of Ang II in nociceptive transmission in the spinal cord, we examined the effect of intrathecal (i.t.) administration of Ang II into mice.ResultsI.t. administration of Ang II produced a behavioral response in mice mainly consisting of biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by Ang II (3 pmol) was dose-dependently inhibited by intraperitoneal injection of morphine (0.1-0.3 mg/kg), suggesting that the behavioral response is related to nociception. The nociceptive behavior was also inhibited dose-dependently by i.t. co-administration of losartan (0.3-3 nmol), an Ang II type 1 (AT1) receptor antagonist, and SB203580 (0.1-1 nmol), a p38 MAPK inhibitor. However, the Ang II type 2 (AT2) receptor antagonist PD123319, the upstream inhibitor of ERK1/2 phosphorylation U0126, and the JNK inhibitor SP600125 had no effect on Ang II-induced nociceptive behavior. Western blot analysis showed that the i.t. injection of Ang II induced phosphorylation of p38 MAPK in the lumbar dorsal spinal cord, which was inhibited by losartan, without affecting ERK1/2 and JNK. Furthermore, we found that AT1 receptor expression was relatively high in the lumbar superficial dorsal horn.ConclusionsOur data show that i.t. administration of Ang II induces nociceptive behavior accompanied by the activation of p38 MAPK signaling mediated through AT1 receptors. This observation indicates that Ang II may act as a neurotransmitter and/or neuromodulator in the spinal transmission of nociceptive information.