Masakazu Nishimura

Comparison of the antioxidant activity of roasted tea with green, oolong, and black teas

Although the antioxidant properties of green, oolong, and black teas have been well studied, antioxidant activity has not been examined in roasted tea. Therefore, in the current studies, we investigated the antioxidant activity of roasted tea in comparison with those of green, oolong, and black teas. Using water extracts of the various teas, we examined the total phenolic content as well as the antioxidant activities, including the reducing power, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, and the inhibition of hemolysis caused by 2,2’-azo-bis(2-amidinopropane) dihydrochloride (AAPH)-induced lipid oxidation in erythrocyte membranes. The roasted tea contained lower levels of total phenolics than green, oolong, or black tea (green tea>oolong tea>black tea>roasted tea). The relative reducing power and DPPH scavenging activity decreased in the following order: green tea>roasted tea>oolong tea>black tea. Also, green tea was more effective against AAPH-induced erythrocyte hemolysis than other teas (green tea>roasted tea=oolong tea=black tea). These results suggest that roasted tea is beneficial to health, in humans, because of its high antioxidant activity.

Volatile female odors activate the accessory olfactory system of male mice without physical contact

We previously reported that male mice are more attracted to volatile odors from intact female mice than from ovariectomized female mice. In the present study, we investigated male attraction to volatile odors from soiled bedding collected from the cages of estrous or ovariectomized female mice. There was no difference in the total time spent sniffing volatile odors from estrous and ovariectomized female mice, suggesting that female mice emit volatile odors which are not excreted into bedding. To test this possibility, we investigated c-Fos expression in the mitral cell layer and granule cell layer of the accessory olfactory bulb 60 min after exposure of male mice to volatile odors without physical contact. Volatile odors from an estrous female mouse significantly increased the total number of c-Fos positive cells in each of the rostral and caudal granule cell layer, but not in the mitral cell layer. After exposure to volatile odors from estrous bedding, the total number of c-Fos positive cells did not increase. Volatile odors from a male mouse did not increase the total number of c-Fos positive cells. Volatile odors from an ovariectomized female mouse increased c-Fos expression only in the caudal granule cell layer. These results suggest that female mice emit specific volatile odors which are not excreted into bedding, and that the volatile odors activate the accessory olfactory system of male mice without physical contact. To characterize the female-specific volatile odors, we conducted habituation-dishabituation tests. Whereas sham-operated male mice discriminated between volatile odors of estrous and ovariectomized female mice, vomeronasal organ-removed male mice did not. These results suggest that male mice discriminated whether or not female mice were ovariectomized, by volatile odors via the accessory olfactory system, and that the female-specific volatile odors are involved in reproduction.

Three distinct muscarinic signalling pathways for cationic channel activation in mouse gut smooth muscle cells

Using mutant mice genetically lacking certain subtypes of muscarinic receptor, we have studied muscarinic signal pathways mediating cationic channel activation in intestinal smooth muscle cells. In cells from M2 subtype‐knockout (M2‐KO) or M3‐KO mice, carbachol (100 μm) evoked a muscarinic cationic current (mICat) as small as ∼10% of mICat in wild‐type (WT) cells. No appreciable current was evoked in M2/M3 double‐KO cells. All mutant type cells preserved normal G‐protein–cationic channel coupling. The M3‐KO and WT mICat each showed a U‐shaped current–voltage (I–V) relationship, whereas the M2‐KO mICat displayed a linear I–V relationship. Channel analysis in outside‐out patches recognized 70‐pS and 120‐pS channels as the major muscarinic cationic channels. Active patches of M2‐KO cells exhibited both 70‐pS and 120‐pS channel activity usually together, either of which consisted of brief openings (the respective mean open times Oτ= 0.55 and 0.23 ms). In contrast, active M3‐KO patches showed only 70‐pS channel activity, which had three open states (Oτ= 0.55, 3.1 and 17.4 ms). In WT patches, besides the M2‐KO and M3‐KO types, another type of channel activity was also observed that consisted of 70‐pS channel openings with four open states (Oτ= 0.62, 2.7, 16.9 and 121.1 ms), and patch current of this channel activity showed a U‐shaped I–V curve similar to the WT mICat. The present results demonstrate that intestinal myocytes are endowed with three distinct muscarinic pathways mediating cationic channel activation and that the M2/M3 pathway targeting 70‐pS channels, serves as the major contributor to mICat generation. The delineation of this pathway is consistent with the formation of a functional unit by the M2‐Go protein and the M3‐PLC systems predicted to control cationic channels.

Effects of extracellular Ca2+ on phagocytosis and intracellular Ca2+ concentrations in polymorphonuclear leukocytes of postpartum dairy cows

The aim of this study was to determine the effects of extracellular Ca(2+) concentration ([Ca(2+)](e)) on phagocytosis and intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine polymorphonuclear leukocytes (PMNs). The experiments were performed by using blood samples from parturient paretic and clinically normal parturient cows and manipulating the [Ca(2+)](e) in vitro. Phagocytosis by PMNs (with and without stimulation with phorbol myristate acetate and inhibition with cytochalasin B) and resting [Ca(2+)](i) were significantly lower in parturient paretic cows. Repletion of Ca(2+) in the extracellular media for the samples from these animals increased phagocytosis and resting [Ca(2+)](i). In the blood of clinically normal parturient cows, decreasing the [Ca(2+)](e) decreased phagocytosis and resting [Ca(2+)](i) in PMNs, but increasing the [Ca(2+)](e) did not affect phagocytosis. These results suggest that the hypocalcemic condition of parturient paretic cows in vivo causes decreased phagocytosis and resting [Ca(2+)](i) in PMNs, which may partly contribute to greater susceptibility to infection.

Palytoxin-induced increase in cytosolic-free Ca2+ in mouse spleen cells

The effect of palytoxin (C(129)H(223)N(3)O(54)) on Ca(2+) homeostasis in immune cells has not been studied. Therefore, we investigated the effect of palytoxin on the cytosolic-free Ca(2+) concentration ([Ca(2+)](i)) in mouse spleen cells using a fluorescence Ca(2+) indicator, fura-2. Palytoxin (0.1-100 nM) increased [Ca(2+)](i) in a concentration-dependent manner. The palytoxin-induced increase in [Ca(2+)](i) was abolished by the omission of extracellular Ca(2+) or 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF-96365, 100 microM), and was greatly inhibited by Ni(2+) (2 mM). Ouabain (0.5-1 mM) partially inhibited the palytoxin-induced response. There was no effect of decreased extracellular Na(+) (6.2 mM), tetrodotoxin (1 microM), verapamil (10 microM), nifedipine (10 microM), omega-agatoxin IVA (200 nM), omega-conotoxin GVIA (1 microM), omega-conotoxin MVIIC (500 nM), or La(3+) (100 microM). These results suggest that palytoxin increases [Ca(2+)](i) in mouse spleen cells by stimulating Ca(2+) entry through an SKF-96365-, Ni(2+)-sensitive pathway.

A competitive effect of androgen signaling on male mouse attraction to volatile female mouse odors.

Olfaction plays an important role in animal communication. We hypothesized that males recognize the attractive volatile odors attributed to female reproductive ability. We measured the period during which a male mouse spent sniffing volatile odors from a sham-operated female mouse or an ovariectomized mouse without visual or tactile contact. Intact male mice spent more time sniffing volatile odors from proestrous, estrous or metestrous females than from ovariectomized females. There was no difference in castrated male mice. To investigate the involvement of sexual hormone in this behavior, castrated male mice were treated with 17 alpha-estradiol (E), dihydrotestosterone (DHT), or both. E-treatment did not affect sniffing behavior. Regardless of the estrous stages, DHT-treated castrated males spent less time sniffing the volatile odors from sham-operated than from ovariectomized female mice. Both E- and DHT-treated castrated males spent less time sniffing the volatile odors from proestrous or estrous females than from ovariectomized females. These results suggest that neither androgen nor estrogen is sufficient for reproducing male attraction to volatile female mouse odors, and that androgen signaling has a competitive effect against the attraction.

Chronic intracerebroventricular administration of anti-neuropeptide Y antibody stimulates starvation-induced feeding via compensatory responses in the hypothalamus.

To investigate how compensatory responses develop after the onset of inhibition of NPY signaling, we examined the effect of continuous intracerebroventricular (ICV) injection of neutralizing NPY antibodies (NPY-ab) on daily and fast-induced food intake in mice. A single ICV injection of NPY-ab reduced food intake in fasted mice. In contrast to a single injection, continuous ICV injection of NPY-ab for 13 days increased fast-induced food intake, although daily food intake was unaffected by continuous administration of NPY-ab. Immunohistochemistry indicated that the expression of NPY protein increases in the arcuate nucleus, lateral hypothalamic area, and paraventricular nucleus 7 days after onset of continuous NPY-ab infusion and remains at an elevated level, whereas the expression of the NPY Y1 receptor transiently increases in the same areas for 3 days and then gradually decreases. Similar results were obtained for the expression of NPY and NPY Y1 receptor mRNA. The mRNA level of agouti-related protein, another orexigenic neuropeptide, also increased in parallel with NPY, whereas that of pro-opiomelanocortin did not change over the 13 days of the NPY-ab administration. These results suggest that chronic central inhibition of NPY immediately activates orexigenic signaling in first-order hypothalamic neurons and that this compensatory mechanism normalizes the regulation of feeding and energy expenditure to maintain energy homeostasis. On the other hand, in mice that have acquired this compensation, fast-induced food intake further increases even after the energy deficit is corrected because of the dominant orexigenic signal.

The mesencephalic trigeminal sensory nucleus is involved in the control of feeding and exploratory behavior in mice.

The mesencephalic trigeminal nucleus (Me5), which receives input from oral proprioceptors and projects to higher brain regions, is involved in mastication-induced modulation of satiation. To investigate how the Me5 is involved in the control of feeding and exploratory behavior, we examined the effect of bilateral electrolytic lesions of the Me5 on feeding and exploratory behavior in mice. Mouse feeding and exploratory behaviors were analyzed using a food-search-compulsion-apparatus (FSCA), which was designed to distinguish between the two behaviors under standard living conditions. To assess anxiety in mice in an unfamiliar environment, exploratory activity was analyzed in an automated hole-board apparatus. Mice with bilateral Me5 lesions had unique feeding and exploratory behavior profiles in the FSCA compared with sham-operated mice. Me5-lesioned mice spent more time in the food chamber during each trial in the FSCA, but the number of entries into the food chamber was decreased by 40% compared to sham-operated mice. Moreover, Me5 lesions markedly inhibited exploratory behavior, manifested as low-frequency exploration. In spite of the low-frequency exploration in the FSCA, Me5 lesions had no effect on various exploratory activities analyzed in the hole-board apparatus, i.e., total locomotor activity, frequency and duration of rearing and head-dipping, and latency to the first head-dipping. These results suggest that the Me5 is involved in the control of feeding and exploratory behavior through its ascending neuronal pathways in mice without modulating the emotional state.

Sex steroids modulate the signals from volatile female odors in the accessory olfactory bulb of male mice

We previously reported that male mice detect volatile female odors via the accessory olfactory system, and that these odors activate granule cells in the accessory olfactory bulb (AOB) with a characteristic pattern. We also reported that sex steroids modulate the attraction of male mice to volatile female odors. The present study investigated hormonal modulation of signals from volatile female odors in the AOB with c-Fos immunostaining. After intact male mice were exposed to volatile female odors, there were more c-Fos positive cells in the caudal granule cell layer (GCL) than in the rostral GCL of the AOB. This effect was observed 3 days but not 7 days after castration, suggesting that hormonal deficiency causes the reorganization of the AOB after 3 days. There was no difference in the number of c-Fos positive cells between the rostral and caudal GCL of castrated male mice treated with 17 beta-estradiol (E). In contrast, there were more c-Fos positive cells in the caudal GCL than in the rostral GCL of castrated male mice treated with dihydrotestosterone (DHT). In both DHT- and E-treated castrated male mice, there was no difference in the number of c-Fos positive cells between the rostral GCL and caudal GCL. This finding suggests that E disrupts the effect of DHT, and that androgen is required for maintaining the intact neuronal network of the AOB. The present study suggests that sex steroids modulate the signals from volatile female odors in the AOB of male mice.

Prevalence of Toxoplasma gondii and Neospora caninum in Sika Deer from Eastern Hokkaido, Japan

Brain and serum were collected from 120 and 12 free-ranging sika deer (Cervus nippon yesoensis), respectively, from six regions in eastern Hokkaido during controlled hunts in the autumn of 2003. Brains were tested for Neospora caninum and Toxoplasma gondii DNA by polymerase chain reaction (PCR) assays. Antibodies to Toxoplasma gondii were measured by means of a latex agglutination test. No brain tested positive for either type of DNA, and no antibody to Toxoplasma gondii was detected in serum, suggesting a low prevalence of infection with these organisms in free-ranging sika deer from eastern Hokkaido. Further examination of multiple tissues by PCR and serologic surveys will be necessary to confirm this.