Molecular mechanisms of large-conductance ca (2+) -activated potassium channel activation by ginseng gintonin.

Gintonin is a unique lysophosphatidic acid (LPA) receptor ligand found in Panax ginseng. Gintonin induces transient [Ca(2+)]i through G protein-coupled LPA receptors. Large-conductance Ca(2+)-activated K(+) (BKCa) channels are expressed in blood vessels and neurons and play important roles in blood vessel relaxation and attenuation of neuronal excitability. BKCa channels are activated by transient [Ca(2+)]i and are regulated by various Ca(2+)-dependent kinases. We investigated the molecular mechanisms of BKCa channel activation by gintonin. BKCa channels are heterologously expressed in Xenopus oocytes. Gintonin treatment induced BKCa channel activation in oocytes expressing the BKCa channel α subunit in a concentration-dependent manner (EC50 = 0.71 ± 0.08 µg/mL). Gintonin-mediated BKCa channel activation was blocked by a PKC inhibitor, calphostin, and by the calmodulin inhibitor, calmidazolium. Site-directed mutations in BKCa channels targeting CaM kinase II or PKC phosphorylation sites but not PKA phosphorylation sites attenuated gintonin action. Mutations in the Ca(2+) bowl and the regulator of K(+) conductance (RCK) site also blocked gintonin action. These results indicate that gintonin-mediated BKCa channel activations are achieved through LPA1 receptor-phospholipase C-IP3-Ca(2+)-PKC-calmodulin-CaM kinase II pathways and calcium binding to the Ca(2+) bowl and RCK domain. Gintonin could be a novel contributor against blood vessel constriction and over-excitation of neurons.

Development of sequencing batch reactor with step feed and recycle.

SBR process shows high nitrogen and phosphorus removal in countries where separated sewers are used. On the other hand, removal efficiency is low in countries where combined sewers are used though the same SBR is applied. This is because the organic concentration (as BOD), which is used as carbon source for denitrification, of combined sewers is much lower than that of separated sewers. Almost all sewers in Korea are combined, and their BOD is low by about 1/2 over the level needed for denitrification. In this study, a SBR process that can optimise organic usage by step feed and recycle is thus developed to increase the removal efficiency of nitrogen and phosphorus, and the results show that the removal rates of BOD, T-N and T-P are 95.4, 81.4 and 86.1%, respectively, though influent BOD is low.

Development of soil-covered SBR process for small scale sewage treatment.

Nitrogen and phosphorus compounds, mainly from the households in residential areas are responsible for eutrophication occurring in lakes or estuaries. In Korea, there is a growing trend for villages in the countryside, rest areas of express ways, military unit bases, etc. to use small scale sewage treatment plants (SSTPs). However, the technological developments have mainly focused on medium or large scale sewage treatment plants, leaving a gap in the development of technologies for SSTPs without a residing operator. In this study, a soil-covered sequencing batch reactor (SBR) process for SSTPs in combination with a natural purification technology is developed to remove nitrogen and phosphorus as well as organic contamination (BOD), and the results are as follows: (1) the removal rates of BOD, total nitrogen (T-N), total phosphorus (T-P) and suspended solids (SS) are 93%, 74%, 80% and 95%, respectively, (2) the soil-covered SBR process without the decanter used for effluent discharge is similar to other SBR processes in suspended solids removal rate, and (3) the performance of the field pilot plant was monitored by remote sensing system in real time, and the results show that this system can be applied to SSTPs with no residing operators.

Neural substrates, experimental evidences and functional hypothesis of acupuncture mechanisms.

OBJECTIVES: Although acupuncture therapy has demonstrated itself to be effective in several clinical areas, the underlying mechanisms of acupuncture in general and the analgesic effect in particular are, however, still not clearly delineated. We, therefore, have studied acupuncture analgesic effect through fMRI and proposed a hypothesis, based on the obtained result, which will enlighten the central role of the brain in acupuncture therapy. METHODS: The proposed model, termed as a broad sense hypothalamus-pituitary-adrenal (BS-HPA) axis, was based on our observed neuroimaging results. The model incorporates the stress-induced HPA axis model together with neuro-immune interaction including the cholinergic anti-inflammatory model. RESULTS: The obtained results coupled with accumulating evidence suggest that the central nervous system is essential for the processing of these effects via its modulation of the autonomic nervous system, neuroimmune system and hormonal regulation. CONCLUSIONS: Based on our fMRI study, it appears that understanding the effects of acupuncture within a neuroscience-based framework is vital. Further, we have proposed the broad sense-HPA axis hypothesis which incorporates the experimental results.

Deafferentation-disconnection neglect induced by posterior cerebral artery infarction.

OBJECTIVE: To investigate patients with posterior cerebral artery (PCA) infarctions to learn whether hemispatial neglect is more frequent and severe after right than left PCA infarction; whether visual field defects (VFDs) influence the presence or severity of hemispatial neglect; and the anatomic loci of lesions that are associated with hemispatial neglect. METHODS: The authors recruited 45 patients with PCA infarction that involved only the occipital lobe or the occipital lobe plus other areas served by the PCA. All subjects received seven neglect tests within 2 months after onset. RESULTS: Overall, the frequency of hemispatial neglect was 42.2%. The frequency did not significantly differ between the right (48.0%) and left (35.0%) PCA groups, but the severity of hemispatial neglect was significantly greater in the right group. VFD alone did not influence the frequency or severity of neglect after controlling other variables. Isolated occipital lesions were rarely associated with hemispatial neglect, and it was only the occipital plus splenial lesion that significantly influenced the frequency and severity of neglect. CONCLUSIONS: This study suggests that after excluding such confounding factors as aphasia or hemiplegia, neglect frequency does not differ between the right and left posterior cerebral artery (PCA) groups, but the severity of neglect is greater after right PCA infarctions; even in the acute stage of PCA infarction; visual field defect from an isolated occipital lesion does not cause hemispatial neglect; and the injury to both the occipital lobe and the splenium of the corpus callosum is important for producing hemispatial neglect with PCA infarction.

Development of a miniaturized assay for the high-throughput screening program for poly(ADP-ribose) polymerase-1.

Poly(ADP-ribose) polymerase (PARP) plays a pivotal role in the repair of DNA strand breaks. However, excessive activation of PARP causes a rapid depletion of intracellular energy, leading to cell death. PARP inhibitors may have potential therapeutic benefit in the treatment of myocardial ischemia, stroke, and neurodegenerative disease. With these emerging medicinal interests, various screening programs have identified small molecules that inhibit PARP with reasonable potencies. However, the increasing numbers of diverse small molecules generated through combinatorial chemistry necessitate the use of robust assays with good sensitivity and specificity for use as a high-throughput screening (HTS) program. Here, we report the development and the validation of a nonisotopic PARP-1 assay suitable for HTS by converting a biotinylated NAD-based colorimetric assay to a miniaturized 384-well plate format. Comparing with the conventional methods, this miniaturized PARP-1 inhibition assay was equally sensitive with excellent reproducibility and cost-effectiveness. Because nonisotopic PARP-1 inhibition assays are widely used, the methodology described in this article can expand the feasibility of this assay as a high-throughput assay for screening of PARP-1 inhibitors from a random chemical library.

Neurophysiological identification and characterization of thalamic neurons with single unit recording in essential tremor patients.

This study investigated the characteristics of the neuronal activities of the motor thalamus (Vim and Vop) in essential tremor (ET) patients, and compared the results with those of Parkinson's disease (PD) patients. The kinetic (Ki) neurons were found mainly in the Vim, whereas the voluntary (Vo) neurons were found principally in the Vop of ET patients. The mean firing rates of the ET patients were higher than those of the PD patients. In addition, the mean firing rates of the Ki neurons of the ET patients were higher than those of the PD patients in the Vim nuclei. However, the mean firing rates of the ventralis caudalis (Vc) neurons, which respond to sensory stimulation, were similar in each group. An analysis of the incidence of bursting neurons revealed that the Vop nucleus of the ET patients had less bursting neurons than the PD patients. However, in the Vim nucleus, both groups possessed bursting neurons even though the incidence was slightly different. Tremor cells were observed less frequently in the Vim nucleus of ET patients than in the PD patients. This study demonstrated the characteristic features of the neuronal activities of ET patients compared to those of PD patients.

Hepatoprotective and free radical scavenging effects of Nelumbo nucifera.

Ethanol extracts from Nelumbo nucifera (ENN) seeds were studied for possible antioxidative and hepatoprotective effects. Antioxidative effects were measured spectrophotometrically by reduction of 2,2'-Diphenyl-1-picrylhydrazyl (DPPH) radicals. Hepatoprotective effects were tested using carbon tetrachloride (CCl4) and aflatoxin B1 (AFB1)-induced hepatocyte toxicity models. ENN showed potent free radical scavenging effects with a median inhibition concentration of 6.49 microg/ml. Treatment of hepatocytes with ENN inhibited both the production of serum enzymes and cytotoxicity by CCl4. The genotoxic and cytotoxic effects of AFB1 were also inhibited by ENN in dose-dependent manners. These hepatoprotective effects of ENN against CCl4 and AFB1 might result from its potent antioxidative properties.

Effects of luteolin on the inhibition of proliferation and induction of apoptosis in human myeloid leukaemia cells.

Luteolin, a flavonoid isolated from the fruit of Vitex rotundifolia, has been examined with regard to the inhibition of proliferation and induction of apoptosis in human myeloid leukaemia HL-60 cells. The concentration required for 50% inhibition of the growth after 96 h was 15 +/- 1.1 microM. The mode of cell death induced by luteolin was found to be apoptosis, as judged by the morphologic alteration of the cells and by the detection of DNA fragmentation using agarose gel electrophoresis. The degree of apoptosis was quantified by a sandwich enzyme immunoassay and flow cytometric analysis. These results suggest that luteolin may be used as potential chemopreventive and chemotherapeutic agents.

AtHKT1 is a salt tolerance determinant that controls Na(+) entry into plant roots.

Two Arabidopsis thaliana extragenic mutations that suppress NaCl hypersensitivity of the sos3-1 mutant were identified in a screen of a T-DNA insertion population in the genetic background of Col-0 gl1 sos3-1. Analysis of the genome sequence in the region flanking the T-DNA left border indicated that sos3-1 hkt1-1 and sos3-1 hkt1-2 plants have allelic mutations in AtHKT1. AtHKT1 mRNA is more abundant in roots than shoots of wild-type plants but is not detected in plants of either mutant, indicating that this gene is inactivated by the mutations. hkt1-1 and hkt1-2 mutations can suppress to an equivalent extent the Na(+) sensitivity of sos3-1 seedlings and reduce the intracellular accumulation of this cytotoxic ion. Moreover, sos3-1 hkt1-1 and sos3-1 hkt1-2 seedlings are able to maintain [K(+)](int) in medium supplemented with NaCl and exhibit a substantially higher intracellular ratio of K(+)/Na(+) than the sos3-1 mutant. Furthermore, the hkt1 mutations abrogate the growth inhibition of the sos3-1 mutant that is caused by K(+) deficiency on culture medium with low Ca(2+) (0.15 mM) and <200 microM K(+). Interestingly, the capacity of hkt1 mutations to suppress the Na(+) hypersensitivity of the sos3-1 mutant is reduced substantially when seedlings are grown in medium with low Ca(2+) (0.15 mM). These results indicate that AtHKT1 is a salt tolerance determinant that controls Na(+) entry and high affinity K(+) uptake. The hkt1 mutations have revealed the existence of another Na(+) influx system(s) whose activity is reduced by high [Ca(2+)](ext).

Rotundifuran, a labdane type diterpene from Vitex rotundifolia, induces apoptosis in human myeloid leukaemia cells.

The inhibitory effect of rotundifuran, a labdane type diterpene isolated from the fruit of Vitex rotundifolia, on the proliferation of human myeloid leukaemia HL-60 cells was examined. The concentration required for 50% inhibition of the growth after 96 h was 22.5 microM. The mode of cell death induced by rotundifuran was found to be apoptosis, which was judged by the morphological alteration of the cells and by the detection of DNA fragmentation using agarose gel electrophoresis. The degree of apoptosis was quantified by a sandwich enzyme immunoassay and flow cytometric analysis. These results suggest that rotundifuran may be used as a potential chemopreventive and chemotherapeutic agent.

Studies of the central neural pathways to the stomach and Zusanli (ST36).

The purpose of this morphological study was to investigate the relation between the meridian, meridian points and viscera using neuroanatomical tracers. The common locations of the spinal cord and brain projecting to the stomach and Zusanli were observed following injection of CTB (cholera toxin B subunit) and pseudorabies viruses (PRV-Ba, Bartha strain and PRV-Ba-Gal, galactosidase insertion) into the stomach and Zusanli (ST36). After 4-5 days of survival following injection into twelve rats, they were perfused, and their spinal cords and brains were frozen sectioned (30 microm). These sections were stained by X-gal histochemical, CTB and PRV-Ba immunohistochemical staining methods, and examined with the light microscope. The results were as follows: Commonly labeled medulla oblongata regions were dorsal motor nucleus of vagus nerve (DMV), nucleus tractus solitarius (NTS) and area postrema (AP) following injection of CTB and PRV-Ba-Gal into stomach and Zusanli, respectively. In the spinal cord, commonly labeled neurons were found in thoracic, lumbar and sacral spinal segments. Densely labeled areas were found in lamina IV, V, VII (intermediolateral nucleus) and X of the spinal cord. In the brain, commonly labeled neurons were found in the Al noradrenalin cells/Cl adrenalin cells/caudoventrolateral reticular nucleus, dorsal motor nucleus of vagus nerve, nucleus tractus solitarius, area postrema, raphe obscurus nucleus, raphe pallidus nucleus, raphe magnus nucleus, gigantocellular nucleus, locus coeruleus, parabrachial nucleus, Kolliker-Fuse nucleus, A5 cell group, central gray matter, paraventricular hypothalamic nucleus, lateral hypothalamic nucleus, retrochiasmatic hypothalamic nucleus, bed nucleus of stria terminalis and amygdaloid nucleus. Thus central autonomic center project both to the stomach and Zusanli. These morphological results suggest that there is a commonality of CNS cell groups in brain controlling stomach (viscera) and Zusanli (limb).

Fumigant toxicity of volatile natural products from Korean spices and medicinal plants towards the rice weevil, Sitophilus oryzae (L).

The fumigant toxicity of various volatile constituents of essential oils extracted from sixteen Korean spices and medicinal plants towards the rice weevil, Sitophilus oryzae L (Coleoptera: Curculionidae), was determined. The most potent toxicity was found in the essential oil from Mentha arvensis L. var piperascens (LC50 = 45.5 microliters litre-1 air). GC-MS analysis of essential oil from M arvensis showed it to be rich in menthol (63.2%), menthone (13.1%) and limonene (1.5%), followed in abundance by beta-pinene (0.7%), alpha-pinene (0.6%) and linalool (0.2%). Treatment of S oryzae with each of these terpenes showed menthone to be most active (LC50 = 12.7 microliters litre-1 air) followed by linalool (LC50 = 39.2 microliters litre-1 air) and alpha-pinene (LC50 = 54.9 microliters litre-1 air). Studies on inhibition of acetylcholinesterase activity of S oryzae showed menthone to have a nine-fold lower inhibitory effect than menthol, despite menthone being 8.1-fold more toxic than menthol to the rice weevil. Different modes of toxicity of these monoterpenes towards S oryzae are discussed.

Effects of Shikunshito-Kamiho on fecal enzymes and formation of aberrant crypt foci induced by 1,2-dimethylhydrazine.

Shikunshito-Kamiho (SKTK) is a traditional Chinese medicine composed of eight crude drugs (Ginseng Radix, Hoelen, Atractylodis Rhizoma, Glycyrrhizae Radix, Prunellae Spica, Ostreae Testa, Laminaria Thallus, Sargassum). We investigated the effects of SKTK on pH, ammonia, fecal enzymes such as beta-glucuronidase, tryptophanase, urease, and formation aberrant crypt foci in the colon carcinogenesis model induced by 1,2-dimethylhydrazine (DMH). Water extract of SKTK was administered orally for 5 weeks to DMH-treated mice as 0.5% and 1.5% of the diet. Beta-glucuronidase, pH and tryptophanase were significantly inhibited after treatment of 0.5% and 1.5% SKTK, while urease was significantly reduced only during and after treatment of 1.5% SKTK as compared with control data. However, the ammonia concentration wasn't different in SKTK treated groups from control group. The incidence number of aberrant crypts foci (ACF) and aberrant crypts/focus in colon was significantly decreased by 0.5% and 1.5% SKTK mixed diets compared with that in rats treated with DMH alone. These results suggest that SKTK exterts anticarcinogenic activity on experimental murine colorectal cancer.

A new antioxidant monoterpene glycoside, alpha-benzoyloxypaeoniflorin from Paeonia suffruticosa.

Alpha-benzoyloxypaeoniflorin (1), a new antioxidant monoterpene alpha-glycoside anomer was isolated from Paeonia suffruticosa along with known compounds, beta-benzoyloxypaeoniflorin (2), paeonolide, paeoniflorin and mudanpioside H. The structure of 1 has been determined by comparing spectral data with those of beta-benzoyloxypaeoniflorin (2). Compound 1 exhibited moderately potent radical scavenging activity on DPPH radical.

Impacts of agrochemical fertilizer on the aquatic environment of paddy fields in Vietnam.

To evaluate the impacts of agrochemical fertilizer application on the aquatic environment of paddy fields in tropical regions, 7.04 ha of paddy field situated in northern Vietnam were selected as a study area. The fate of nutrient constituents was surveyed through a questionnaire as well as analytical observation. Taking the major environmental components of the paddy field into account, a mass-balance flow regarding fertilizing constituents such as nitrogen and phosphate was built up and used to estimate the contribution of fertilizer to paddy field water pollution. In the mass-flow analysis, the randomness of fertilizer used by local farmers is incorporated within the conventional input-output model. For the control volume of soil in 0-40 cm below paddy plot surface, the estimated average concentrations in soil were 1.8 ppm for inorganic nitrogen and 1.3 ppm for inorganic phosphate on the assumption of 50% non-excessive probability. From the reasonable consistency of the deduced concentrations of nutrients with observed ones, not only the mass balance of fertilizing substances but also the impacts of fertilizer on the paddy field aquatic environment in Vietnam could be understood. On the results from nutrient mass-balance analysis, it was found that 11.3-13.3 kg N/ha would percolate into the underground aquifer that is linked to surface channel flow in the vicinity of the paddy plots and affect the irrigation water quality. The nutrient portion in the materials harvested as grain and straw occupied 58% of total dosed nitrogen and 75.6% of total supplied phosphorous.

Effect of supplementation of vitamin E and vitamin C on brain acetylcholinesterase activity and neurotransmitter levels in rats treated with scopolamine, an inducer of dementia.

In the present study, the effects of vitamins E and C on the levels of neurotransmitters and acetylcholinesterase activity in the brains of rats treated with scopolamine, an inducer of dementia, were examined. Fifty male Sprague-Dawley rats at the age of 5 wk were divided into five groups after 1 wk of adaptation and fed five different diets for 6 wk: a no-scopolamine group, which was a scopolamine-untreated group fed only a basal diet: a scopolamine-treated group fed a basal diet; a vitamin E-supplemented scopolamine-treated group: a vitamin C-supplemented scopolamine-treated group; and a vitamins E and C-supplemented scopolamine-treated group. Scopolamine was twice administered by intraperitoneal injection (300 mg/kg, body weight), 3 d and 20 min prior to sacrifice. Brain acetylcholinesterase activity was markedly reduced by scopolamine injection. However, the supplementation of vitamins E and C in the diet significantly increased the reduced brain acetylcholinesterase activity up to the level of the scopolamine-untreated group. Brain serotonin concentration in the vitamin C-supplemented scopolamine-treated group was significantly higher than that in the scopolamine-treated group. However, there were no significant differences in brain dopamine and norepinephrine concentrations among all groups. In conclusion, supplementation with vitamin E and/or vitamin C might be useful in maintaining brain acetylcholinesterase activity at the normal level and serotonin concentration for some extent under the condition to induce dementia by scopolamine administration.

Polymethoxyflavonoids from Vitex rotundifolia inhibit proliferation by inducing apoptosis in human myeloid leukemia cells.

Three polymethoxyflavonoids from the fruit of Vitex rotundifolia, namely 2',3',5-trihydroxy-3,6,7-trimethoxyflavone (Vx-1), vitexicarpin (Vx-5) and artemetin (Vx-6), were tested for their antiproliferative activity in human myeloid leukemia HL-60 cells. They showed a dose-dependent decrease in the growth of HL-60 cells. The concentrations required for 50% inhibition of the growth (IC50) after 96 h were 4.03 microM, 0.12 microM and 30.98 microM for Vx-1, Vx-5 and Vx-6, respectively. Treatment of HL-60 cells with the flavonoids induced morphological changes that are characteristic of apoptosis. We judged the induction of apoptosis by the detection of DNA fragmentation in agarose gel electrophoresis and the degree of apoptosis was quantified by a double-antibody sandwich ELISA and by flow cytometric analysis. The C-3 hydroxyl and C-8 methoxyl groups were found not to be essential for the activity, but the C-3' methoxyl instead of hydroxyl group lowered the antiproliferative and apoptosis inducing activity. These results suggest that the polymethoxyflavonoids isolated from V. rotundifolia may be used as potential chemopreventive and chemotherapeutic agents.

The effect of C677T mutation of methylene tetrahydrofolate reductase gene and plasma folate level on hyperhomocysteinemia in patients with meningomyelocele.

To evaluate the relationship between genotypes of methylene tetrahydrofolate reductase (MTHFR), and plasma folate and homocysteine (Hcy) levels in meningomyelocele, 21 Korean patients, 47 of their family members, and 43 healthy controls were recruited. The presence of C677T mutation in the MTHFR gene and plasma concentrations of folate/Hcy were investigated. The genotype frequency of C677T mutation was not higher in study groups (patients and family members). The plasma folate concentration showed no difference either between the study and the control groups or among MTHFR-genotypic groups. The plasma Hcy concentration in homozygotes in the study group was higher than that in the control group, and higher than that in heterozygotes when plasma folate levels were low (P=0.006). Although neither MTHFR genotype nor plasma folate/Hcy level plays a definite part on its own, they seem to have an additive effect on the occurrence of meningomyelocele. Our results support folate supplementation for the prevention of hyperhomocysteinemia and meningomyelocele.