Reactive oxygen species produced by Zn2+ influx after exposure to AMPA, but not NMDA and their capturing effect on nigral dopaminergic protection.

Glutamate excitotoxicity is involved in dopaminergic degeneration in the substantia nigra pars compacta (SNpc). Here we compared vulnerability to neurodegeneration after exposure to NMDA and AMPA. Apomorphine-induced movement disorder and dopaminergic degeneration in the SNpc, which are associated with Parkinson's syndrome, were induced after injection of AMPA into the SNpc of rats, but not after injection of NMDA. Co-injection of 1-naphthyl acetyl spermine (NASPM), a selective blocker of Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptors rescued dopaminergic degeneration and increase in intracellular Zn2+ by AMPA. Furthermore, we tested the effect of capturing reactive oxygen species (ROS) produced by Zn2+ on neuroprotection in vivo. The levels of ROS, which were determined by HYDROP, a membrane-permeable H2O2 fluorescence probe and Aminophenyl Fluorescein (APF), a fluorescence probe for hydroxyl radical and peroxynitrite, were increased after injection of AMPA, but not after co-injection of CaEDTA, an extracellular Zn2+ chelator, suggesting that increase in Zn2+ influx by AMPA elevates the levels of intracellular ROS. AMPA-mediated dopaminergic degeneration was completely rescued by co-injection of either HYDROP or APF. The present study indicates that neurotoxic signaling of the influx of extracellular Zn2+ through Zn2+-permeable GluR2-lacking AMPA receptors is converted to ROS production and that capturing the ROS completely protects dopaminergic degeneration after exposure to AMPA, but not NMDA. It is likely that regulation of the conversion from Zn2+ influx into ROS production plays a key role to preventing Parkinson's syndrome.

Chemical identification of an active component and putative neural mechanism for repellent effect of a native ant's odor on invasive species.

The invasive Argentine ants (Linepithema humile) and the red imported fire ants (Solenopsis invicta) constitute a worldwide threat, causing severe disruption to ecological systems and harming human welfare. In view of the limited success of current pest control measures, we propose here to employ repellents as means to mitigate the effect of these species. We demonstrate that cuticular hydrocarbons (CHCs) used as nestmate-recognition pheromone in the Japanese carpenter ant (Camponotus japonicus), and particularly its (Z)-9-tricosene component, induced vigorous olfactory response and intense aversion in these invasive species. (Z)-9-Tricosene, when given to their antennae, caused indiscriminate glomerular activation of antennal lobe (AL) regions, creating neural disarray and leading to aversive behavior. Considering the putative massive central neural effect, we suggest that the appropriate use of certain CHCs of native ants can facilitate aversive withdrawal of invasive ants.

Preventive effect of Ninjin-yoei-to, a Kampo medicine, on amyloid ß1-42-induced neurodegeneration via intracellular Zn2+ toxicity in the dentate gyrus.

Ninjin-yoei-to (NYT), a Kampo medicine, has ameliorative effects on cognitive dysfunction via enhancing cholinergic neuron activity. To explore an efficacy of NYT administration for prevention and cure of Alzheimer's disease, here we examined the effect of NYT on amyloid ß1-42 (Aß1-42)-induced neurodegeneration in the dentate gyrus. A diet containing 3% NYT was administered to mice for 2 weeks and human Aß1-42 was intracerebroventricularly injected. Neurodegeneration in the dentate granule cell layer of the hippocampus, which was determined 2 weeks after the injection, was rescued by administration of the diet for 4 weeks. Aß staining (uptake) was not modified in the dentate granule cell layer by pre-administration of the diet for 2 weeks, while Aß1-42-induced increase in intracellular Zn2+ was reduced, suggesting that pre-administration of NYT prior to Aß injection is effective for reducing Aß1-42-induced Zn2+ toxicity in the dentate gyrus. As a matter of fact, Aß1-42-induced neurodegeneration in the dentate gyrus was rescued by pre-administration of NYT. Interestingly, the level of metallothioneins, intracellular Zn2+-binding proteins, which can capture Zn2+ from Zn-Aß1-42 complexes, was elevated in the dentate granule cell layer by pre-administration of NYT. The present study suggests that pre-administration of NYT prevents Aß1-42-mediated neurodegeneration in the dentate gyurs by induced synthesis of metallothioneins, which reduces intracellular Zn2+ toxicity induced by Aß1-42.

Environmental DNA detection of an invasive ant species (Linepithema humile) from soil samples.

Alien ant species (Formicidae, Hymenoptera) cause serious damage worldwide. Early detection of invasion and rapid management are significant for controlling these species. However, these attempts are sometimes hindered by the need for direct detection techniques, such as capture, visual observation, or morphological identification. In this study, we demonstrated that environmental DNA (eDNA) analysis can be used as a monitoring tool for alien ants using Linepithema humile (Argentine ant), one of the most invasive ants, as a model species. We designed a new real-time PCR assay specific to L. humile and successfully detected eDNA from the surface soil. The reliability of eDNA analysis was substantiated by comparing eDNA detection results with traditional survey results. Additionally, we examined the relationship between eDNA concentration and distance from nests and trails. Our results support the effectiveness of eDNA for alien ant monitoring and suggest that this new method could improve our ability to detect invasive ant species.

Age-related vulnerability to nigral dopaminergic degeneration in rats via Zn2+-permeable GluR2-lacking AMPA receptor activation.

On the basis of the evidence that extracellular Zn2+ influx induced with AMPA causes Parkinson's syndrome in rats that apomorphine-induced movement disorder emerges, here we used a low dose of AMPA, which does not increase intracellular Zn2+ level in the substantia nigra pars compacta (SNpc) of young adult rats, and tested whether intracellular Zn2+ dysregulation induced with AMPA is accelerated in the SNpc of aged rats, resulting in age-related vulnerability to Parkinson's syndrome. When AMPA (1 mM) was injected at the rate of 0.05 µl/min for 20 min into the SNpc, intracellular Zn2+ level was increased in the SNpc of aged rats followed by increase in turning behavior in response to apomorphine and nigral dopaminergic degeneration. In contrast, young adult rats do not show movement disorder and nigral dopaminergic degeneration, in addition to no increase in intracellular Zn2+. In aged rats, movement disorder and nigral dopaminergic degeneration were rescued by co-injection of either extracellular (CaEDTA) or intracellular (ZnAF-2DA) Zn2+ chelators. 1-Naphthyl acetyl spermine (NASPM), a selective blocker of Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptors blocked increase in intracellular Zn2+ in the SNpc of aged rats followed by rescuing nigral dopaminergic degeneration. The present study indicates that intracellular Zn2+ dysregulation is accelerated by Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptor activation in the SNpc of aged rats, resulting in age-related vulnerability to Parkinson's syndrome.

Physiological Roles of Bradykinin and Involvement of Bradykinin B2 Receptor in Urethral Function in Humans and Animals.

OBJECTIVE: We investigated the role of bradykinin in urethral function by examining contractile responses in urethral smooth muscle strips isolated from humans and the intraurethral pressure in rats and dogs. METHODS: The contractile responses of human urethral tissue for bradykinin (0.01-10 µmol/L) were examined, and changes in intraurethral pressure induced by bradykinin (0.003-10 µg/kg) in anesthetized rats or dogs were measured. In addition, the effects of pretreatment with the bradykinin B2 receptor antagonist FK3657 were also examined. RESULTS: In smooth muscle strips obtained from human urethra, bradykinin induced contraction, which was inhibited by FK3657 in a concentration-dependent manner. In anesthetized rats and dogs, intravenously administered bradykinin dose-dependently increased intraurethral pressure. FK3657 shifted the intraurethral pressure dose-response curve for bradykinin to the right in rats. The bradykinin-induced elevation of intraurethral pressure was also dose-dependently inhibited by FK3657 in dogs. CONCLUSIONS: The present study provides evidence that bradykinin elicits urethral smooth muscle contraction via the bradykinin B2 receptor, suggesting the potential utility of this receptor as a novel target for the treatment of voiding dysfunction.

FR177995, a novel vacuolar ATPase inhibitor, exerts not only an inhibitory effect on bone destruction but also anti-immunoinflammatory effects in adjuvant-induced arthritic rats.

There is considerable evidence that osteoclasts are involved in the pathogenesis of juxta-articular bone destruction in rheumatoid arthritis. Vacuolar ATPases (V-ATPases), which are highly expressed in the ruffled border membrane of osteoclasts, play a central role in the process of bone resorption, and V-ATPase inhibitors are effective in preventing bone destruction in several animal models of lytic bone diseases. Here, we evaluated for the first time the effects of V-ATPase inhibition in rats with adjuvant-induced arthritis (AIA) using FR177995, a novel V-ATPase inhibitor. FR177995 completely inhibited H(+) transport driven by V-ATPase, but exerted no effect on the H(+) transport activities of F- and P-ATPase, indicating that FR177995 is a specific inhibitor of V-ATPase. FR177995 acted directly on osteoclastic bone resorption and equally inhibited in vitro bone resorption stimulated by IL-1, IL-6 or PTH. In addition, FR177995 dose-dependently reduced retinoic acid-induced hypercalcemia in thyroparathyroidectomized-ovariectomized rats. When FR177995 was administered to AIA rats once a day, the loss of femoral bone mineral density was significantly improved. Moreover, indicators of cartilage damage (arthritis score and glycosaminoglycan content in the femoral condyles) and inflammation parameters (paw swelling volume, erythrocyte sedimentation rate and plasma sialic acid level) were found to be unexpectedly ameliorated. These results strongly suggest that V-ATPase may be an interesting drug target in the treatment of rheumatoid arthritis.

Response of hippocampal mossy fiber zinc to excessive glutamate release.

The response of hippocampal mossy fiber zinc to excessive glutamate release was examined to understand the role of the zinc in excessive excitation in the hippocampus. Extracellular zinc and glutamate concentrations during excessive stimulation with high K(+) were compared between the hippocampal CA3 and CA1 by the in vivo microdialysis. Zinc concentration in the CA3 was more increased than that in the CA1, while glutamate concentration in the CA3 was less increased than that in the CA1. It is likely that more increase in extracellular zinc is linked with less increase in extracellular glutamate in the CA3. To see zinc action in mossy fiber synapses during excessive excitation, furthermore, 1mM glutamate was regionally delivered to the stratum lucidum in the presence of zinc or CaEDTA, a membrane-impermeable zinc chelator, and intracellular calcium signal was measured in the CA3 pyramidal cell layer. The persistent increase in calcium signal during stimulation with glutamate was significantly attenuated in the presence of 100 microM zinc, while significantly enhanced in the presence of 1mM CaEDTA. These results suggest that zinc released from mossy fibers attenuates the increase in intracellular calcium signal in mossy fiber synapses and postsynaptic CA3 neurons after excessive inputs to dentate granular cells.

Zinc release from Schaffer collaterals and its significance.

On the basis of the evidence that approximately 45% of Schaffer collateral boutons are zinc-positive, zinc release from Schaffer collaterals and its action were examined in hippocampal slices. When zinc release from Schaffer collaterals was examined using ZnAF-2, a membrane-impermeable zinc indicator, ZnAF-2 signal in the stratum radiatum of the CA1 was increased by tetanic stimuli at 100 Hz for 1s, suggesting that zinc is released from Schaffer collaterals in a calcium- and impulse-dependent manner. An in vivo microdialysis experiment indicated that the perfusion with 10 microM zinc significantly decreases extracellular glutamate concentration in the CA1. When tetanic stimuli at 100 Hz for 5s were delivered to the dentate granule cells, the increase in calcium signal in the stratum radiatum of the CA1, as well as in the stratum lucidum of the CA3, was attenuated by addition of 10 microM zinc, while enhanced by addition of 1mM CaEDTA, a membrane-impermeable zinc chelator. The increase in calcium signal in the CA1, in which Schaffer collateral synapses exist, during delivery of tetanic stimuli at 100 Hz for 1s to the Schaffer collateral-commissural pathway was also significantly enhanced by addition of 1mM CaEDTA. These results suggest that zinc released from Schaffer collaterals suppressively modulates presynaptic and postsynaptic calcium signaling in the CA1, followed by the suppression of glutamate release.

Characterization of two isoforms of mouse 3(17)alpha-hydroxysteroid dehydrogenases of the aldo-keto reductase family.

Mouse kidney contains two 3(17)alpha-hydroxysteroid dehydrogenases (HSDs) that show essentially the same properties except for their isoelectric points. However, the structural differences and physiological roles of the two enzymes remain unknown. In this study, we have isolated cDNAs for the two 3(17)alpha-HSDs from a total RNA sample of mouse kidney by reverse transcription-PCR. The identity of the cDNAs was confirmed by characterization of the recombinant enzymes that showed the same molecular weights, pI values, pH optima, substrate specificity and inhibitor sensitivity as those of the enzymes from mouse kidney. We also found that the recombinant enzymes reduce precursors of neuroactive progesterone derivatives, 5alpha-dihydrotestoserone, deoxycorticosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate and estrone at low Km values of 0.3-2 microM. The two enzymes belonged to the aldo-keto reductase (AKR) family, and their 323-amino acid sequences differed only by five amino acids. The sequences of the two isoforms are identical to those of proteins that are predicted to be encoded in a gene for AKR1C21 in the database of the mouse genome. However, the mRNAs for the two isoforms were expressed in mouse kidney and other tissues, in which their expression levels were different. The results indicate an important role of 3(17)alpha-HSD in controlling the concentrations of various steroid hormones in the mouse tissues, and suggest the existence of two genes for the two isoforms of the enzyme.

Pharmacological characteristics of a novel nonthiazolidinedione insulin sensitizer, FK614.

We evaluated antidiabetic effects of 3-(2,4-dichlorobenzyl)-2-methyl-N-(pentylsulfonyl)-3 H-benzimidazole-5-carboxamide (FK614), a benzimidazole derivative without a thiazolidinedione structure, which was obtained using C57BL/KsJ-db/db mice (db/db mice). In db/db mice, the potency of FK614 for hypoglycemic effect was comparable to that of rosiglitazone and approximately 15-fold greater than that of pioglitazone. FK614 also showed a potent attenuating effect on hypertriglyceridemia in db/db mice, as well as rosiglitazone and pioglitazone. In C57BL/6J-ob/ob mice (ob/ob mice), ED(50) values of FK614 and pioglitazone for hypoinsulinemic effect were 1.3 and 11.8 mg/kg, respectively. FK614 also improved the impaired glucose tolerance in ob/ob mice. In normal rats, FK614 did not influence plasma glucose and insulin levels but significantly decreased both plasma triglyceride and nonesterified fatty acid levels. FK614 was found to activate peroxisome proliferator-activated receptor (PPAR)gamma-mediated transcriptional activity in the reporter gene assay as well as thiazolidinedione derivatives, although its maximum effect was less than that of thiazolidinedione derivatives. In rat toxicity studies, hemodilution effects for FK614 were less than that for rosiglitazone. Overall, these studies suggest that FK614 improves insulin resistance in such animal models through activation of PPARgamma-mediated transcriptional activity and that it would be a new therapeutic candidate with potential for the treatment of type 2 diabetic patients.

Release of amino acids by zinc in the hippocampus.

Zinc exists in the synaptic vesicles of hippocampal mossy fibers in high concentrations. On the basis of inhibitory zinc action against glutamate release in the hippocampus, the role of zinc in release of several amino acids were studied in rat hippocampus by using in vivo microdialysis. When the hippocampal CA3 region was perfused with 10 microM ZnCl(2), the concentrations of glutamine, serine, arginine, aspartate, and glycine in the perfusate were significantly increased, whereas the concentrations of amino acids except for glycine were not increased by perfusion with 30 microM ZnCl(2). Chelation of endogenous zinc with 50 microM CaEDTA significantly decreased the concentrations of amino acids in the perfusate except for glycine. In the CA1 region, on the other hand, the concentrations of these five amino acids were not increased by perfusion with 10 microM ZnCl(2) and the concentrations of glutamine and glycine were decreased significantly. The present study suggests that zinc enhances release of glutamine, serine, arginine, and aspartate in the CA3 region and attenuates release of glutamine and glycine in the CA1 region. Zinc seems to modulate glutamatergic synapses multifunctionally in the hippocampus, because glutamine, serine, aspartate, and glycine are involved in synaptic neurotransmission.

Mite vectors (Acari: Trombiculidae) of scrub typhus in a new endemic area in northern Kyoto, Japan.

Between 1983 and 1999, 27 human cases of scrub typhus (two fatal) occurred in the Nodagawa River basin of northern Kyoto, Japan, an area where no cases had been previously reported. Antibody screening of infected patients' sera showed that nine of 15 patients had high titers against the Gilliam type of Orientia tsutsugamushi (Hayashi). To determine the vector mite transmitting the disease, we studied rodent and chigger populations in and near a rice field alongside the Nodagawa River between 1996 and 1999. The most common rodent species was Microtus montebelli (Milne-Edwards), representing 73.3% (33/45) of the population. The mite index (average number of mites per infested host) was highest (190.8) in Leptotrombidium pallidum Nagayo, Mitamura & Tamiya parasitizing on M. montebelli, followed by Leptotrombidium intermedium (Nagayo, Mitamura & Tamiya) (174.9) on the same host species. Orientia tsutsugamushi was isolated from 60.5% (23/38) of rodents and from 71.2% (37/52) of pools of engorged L. pallidum. The Gilliam type of O. tsutsugamushi was most prevalent in rodents, and in engorged L. pallidum and it was the only type recovered from 10 isolates inoculated into L 929 cells for indirect immunofluorescence examination. Orientia tsutsugamushi infected 14.3% (181/1263) and 14.8% (306/2066) of engorged and unfed L. pallidum larvae, respectively, and was also detected in 0.055% (2/3634) of unfed L. intermedium, although previous studies suggest that this mite rarely bites humans. These results show that L. pallidum is the primary vector species of scrub typhus in this new endemic area in Japan.

Cellular heterogeneity in long-term surviving cells isolated from eutopic endometrial, ovarian endometrioma and adenomyosis tissues.

Human endometrial tissues regenerate easily after menstruation and childbirth, suggesting the existence of endometrial stem-like cells that can survive and proliferate from a single cell over a long time. To clarify this hypothesis, limiting dilution cultures performed with eutopic endometrial, ovarian endometrioma and adenomyosis cells obtained from a patient, achieved cloning efficiencies of 13.0, 5.0, and 0.8%, respectively. These monoclonal cells survived for more than 24 months. More than 4 types of monoclonal cells were established from eutopic endometrial cells and microscopically were distinctly different from each other. Intraperitoneal injections of dispersed human eutopic endometrial cells did not cause any endometriosis-like lesions in scid mice, but those of endometrial tissue fragments did. IL-6, TNF-alpha, IL-1beta, M-CSF and HGF failed to enhance transplantation of dispersed endometrial cells to the mice. These results indicate that several types of eutopic endometrial cells survive long-term, and that simple regurgitation of eutopic endometrial stem-like cells may not induce peritoneal endometriosis.

Leptin inhibits decidualization and enhances cell viability of normal human endometrial stromal cells.

Recent reports have demonstrated that the peritoneal fluid and serum concentrations of leptin are increased in women with endometriosis. However, the pathophysiological roles of leptin in endometriosis have not been well characterized. In this study, we examined the direct effects of leptin on normal human endometrial stromal cells using an in vitro decidualization assay system with 8-Br-cAMP, a decidualization inducer. No effects of leptin on cell viability and prolactin secretion were found in unstimulated endometrial stromal cells. Leptin dose-dependently enhanced the viability of stromal cells co-stimulated with 8-Br-cAMP and leptin while PRL secretion from the cells was significantly inhibited in a dose-dependent manner. As for 8-Br-cAMP-stimulated cells, leptin significantly enhanced their cell viability in a dose-dependent manner but not their PRL secretion. These results indicate that leptin enhances the cell viability of PRL-non-secreting 8-Br-cAMP-stimulated stromal cells, and that it inhibits the decidualization process of endometrial stromal cells. Increased leptin in endometriotic patients might play an antiapoptotic role in some activated ESCs in the peritoneal cavity to stimulate endometrial cell implantation, and might cause infertility by inhibiting stromal decidualization.

Leukemia inhibitory factor regulates cell survival of normal human endometrial stromal cells.

Leukemia inhibitory factor (LIF) is produced locally in decidual tissues, but its direct effects on endometrial stromal cells have not been well characterized. In this study, we examined the direct effects of LIF on normal human endometrial stromal cells using an in vitro decidualization assay system with 8-Br-cAMP, a decidualization inducer. We found no effects of LIF on cell viability and prolactin secretion of unstimulated endometrial stromal cells. LIF dose-dependently enhanced cell viability, but not prolactin secretion of 8-Br-cAMP-stimulated cells. LIF dose-dependently enhanced the viability of stromal cells co-stimulated with 8-Br-cAMP and LIF without any significant effect on PRL secretion from the cells. Further, the extracellular matrix did not affect these stimulatory effects of LIF on cell viability of 8-Br-cAMP-stimulated endometrial stromal cells. These results indicate that LIF enhances the cell viability of PRL-non-secreting 8-Br-cAMP-stimulated stromal cells, and that the cell survival signals generated by LIF are independent of those generated by the extracellular matrix. LIF produced locally in decidual tissues may enhance cell viability in certain activated endometrial stromal cells in an autocrine or paracrine manner, and possibly protect against cell damage during embryo implantation and trophoblastic invasion.

Local erythematous dermatitis after intravenous docetaxel.

We report the case of a patient who developed a local skin eruption over the vein following intravenous infusion of docetaxel at 4-week intervals. The skin lesions were limited to an area of the forearm proximal to the site of infusion. They appeared 1 week after the infusion and were diagnosed as erythema multiforme (mixed-type) based on the histological findings.