Serine/threonine kinase 31 promotes PDCD5-mediated apoptosis in p53-dependent human colon cancer cells.

Although programed cell death 5 (PDCD5) is an important protein in p53-mediated proapoptotic signaling, very little is known about PDCD5-related cell death. In this study, we report that serine/threonine kinase 31 (STK31) interacts with PDCD5, which maintains the stability of PDCD5. STK31 overexpression significantly activated PDCD5 stabilization and p53-mediated apoptosis in response to etoposide (ET). However, STK31 knockdown did not enhance apoptosis by ET treatment. Moreover, when STK31 was depleted, PDCD5 inhibited the activation of the p53 signaling pathway with ET, indicating that the PDCD5-STK31 network has an essential role in p53 activation. Importantly, STK31 activated the p53 signaling pathway by genotoxic stress through positive regulation of PDCD5-mediated apoptosis. We thus demonstrated that overexpression of STK31 greatly inhibited tumorigenic growth and increased the chemosensitivity of HCT116 human colorectal carcinoma cells. Taken together, these findings demonstrate that the STK31-PDCD5 complex network regulates apoptosis of cancer cells, and STK31 is a positive apoptosis regulator that inhibits tumorigenesis of colon cancer cells by inducing PDCD5-mediated apoptosis in response to genotoxic stress.

In vivo assay of the potential gadolinium-induced toxicity for sensory hair cells using a zebrafish animal model.

Recently, intratympanic injection of gadolinium-based contrast agent (GdC) is growing in use to visualize the endolymphatic hydrops. Although GdC has been quite safely used over 20 years through intravenous injection, the biological influence of GdC on sensory hair cells needs to be thoroughly assessed for wider clinical application of it through intratympanic injection. In this in vivo experimental study, the summated number of sensory hair cells (SO1, SO2, O1 and OC1 neuromasts) showed a steep decrease in the group exposed to 10% and 20% GdC (35.7 ± 7.3, 15.09 ± 10.82, respectively, P < .01) compared with the control group (47.18 ± 2.30). An increase in apoptosis was also observed in the group exposed to 20% gadolinium (7.20 ± 5.56), as compared with the control group (0.08 ± 0.72) or the group exposed to 10% gadolinium (3.48 ± 3.32). A significant reduction in the viable cytoplasmic mitochondria was observed in embryos exposed to 20% GdC (369 ± 124 µm2 , P = .01) as compared with control embryos (447 ± 118 µm2 ) or embryos exposed to 10% GdC (420 ± 108 µm2 ). GdC administration did not impact peripheral neural structures. GdC caused a significant reduction in sensory hair cell counts in response to high concentrations along with increased apoptosis and mitochondrial damage. However, it may not be likely that GdC will lead to hair cell toxicity, as the estimated concentration in the inner ear after clinically tried intratympanic injection is far more diluted than the non-toxic concentration (0.625%) that was tested in this study.

Enhancement of Seed Dehiscence by Seed Treatment with Talaromyces flavus GG01 and GG04 in Ginseng (Panax ginseng).

Seed dehiscence of ginseng (Panax ginseng C. A. Mayer) is affected by moisture, temperature, storage conditions and microbes. Several microbes were isolated from completely dehisced seed coat of ginseng cultivars, Chunpoong and Younpoong at Gumsan, Korea. We investigated the potential of five Talaromyces flavus isolates from the dehiscence of ginseng seed in four traditional stratification facilities. The isolates showed antagonistic activities against fungal plant pathogens, such as Cylindrocarpon destructans, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia nivalis, Botrytis cinerea, and Phytophthora capsici. The dehiscence ratios of ginseng seed increased more than 33% by treatment of T. flavus GG01, GG02, GG04, GG12, and GG23 in comparison to control (28%). Among the treatments, the reformulating treatment of T. flavus isolates GG01 and GG04 showed the highest of stratification ratio of ginseng seed. After 16 weeks, the reformulating treatment of T. flavus isolates GG01 and GG04 significantly enhanced dehiscence of ginseng seed by about 81% compared to the untreated control. The candidate's treatment of T. flavus GG01 and GG04 showed the highest decreasing rate of 93% in seed coat hardness for 112 days in dehiscence period. The results suggested that the pre-inoculation of T. flavus GG01 and GG04 found to be very effective applications in improving dehiscence and germination of ginseng seed.

Effect of Aerated Compost Tea on the Growth Promotion of Lettuce, Soybean, and Sweet Corn in Organic Cultivation.

This study investigated the chemical characteristics and microbial population during incubation of four kinds of aerated compost teas based on oriental medicinal herbs compost, vermicompost, rice straw compost, and mixtures of three composts (MOVR). It aimed to determine the effects of the aerated compost tea (ACT) based on MOVR on the growth promotion of red leaf lettuce, soybean and sweet corn. Findings showed that the pH level and EC of the compost tea slightly increased based on the incubation time except for rice straw compost tea. All compost teas except for oriental medicinal herbs and rice straw compost tea contained more NO(-) 3-N than NH(+) 4-N. Plate counts of bacteria and fungi were significantly higher than the initial compost in ACT. Microbial communities of all ACT were predominantly bacteria. The dominant bacterial genera were analyzed as Bacillus (63.0%), Ochrobactrum (13.0%), Spingomonas (6.0%) and uncultured bacterium (4.0%) by 16S rDNA analysis. The effect of four concentrations, 0.1%, 0.2%, 0.4% and 0.8% MOVR on the growth of red leaf lettuce, soybean and sweet corn was also studied in the greenhouse. The red leaf lettuce with 0.4% MOVR had the most effective concentration on growth parameters in foliage part. However, 0.8% MOVR significantly promoted the growth of root and shoot of both soybean and sweet corn. The soybean treated with higher MOVR concentration was more effective in increasing the root nodule formation by 7.25 times than in the lower MOVR concentrations Results indicated that ACT could be used as liquid nutrient fertilizer with active microorganisms for culture of variable crops under organic farming condition.

Evaluation of Watermelon Germplasm for Resistance to Phytophthora Blight Caused by Phytophthora capsici.

This study was conducted to determine the Phytophthora rot resistance of 514 accessions of watermelon germplasm, Citrullus lanatus var lanatus. About 46% of the 514 accessions tested were collections from Uzbekistan, Turkey, China, U.S.A., and Ukraine. Phytophthora capsici was inoculated to 45-day-old watermelon seedlings by drenching with 5 ml of sporangial suspension (10(6) sporangia/ml). At 7 days after inoculation, 21 accessions showed no disease symptoms while 291 accessions of susceptible watermelon germplasm showed more than 60.1% disease severity. A total of 510 accessions of watermelon germplasm showed significant disease symptoms and were rated as susceptible to highly susceptible 35 days after inoculation. The highly susceptible watermelon germplasm exhibited white fungal hyphae on the lesion or damping off with water-soaked and browning symptoms. One accession (IT032840) showed moderate resistance and two accessions (IT185446 and IT187904) were resistant to P. capsici. Results suggest that these two resistant germplasm can be used as a rootstock and as a source of resistance in breeding resistant watermelon varieties against Phytophthora.

Chloroplast-targeted expression of synthetic cry1Ac in transgenic rice as an alternative strategy for increased pest protection.

To increase insect resistance in transgenic rice plants, a synthetic truncated cry1Ac gene was linked to the rice rbcS promoter and its transit peptide sequence (tp) for chloroplast-targeted expression. Several transgenic lines were generated by the Agrobacterium-mediated transformation method and the expression levels of the transgene were compared with untargeted expression. Use of the rbcS-tp sequence increased the cry1Ac transcript and protein levels by 25- and 100-fold, respectively, with the accumulated protein in chloroplasts comprising up to 2% of the total soluble proteins. The high level of cry1Ac expression resulted in high levels of plant resistance to three common rice pests, rice leaf folder, rice green caterpillar, and rice skipper, as evidenced by insect feeding assays. Transgenic plants were also evaluated for resistance to natural infestations by rice leaf folder under field conditions. Throughout the entire period of plant growth, the transgenic plants showed no symptoms of damage, whereas nontransgenic control plants were severely damaged by rice leaf folders. Our results demonstrate that the targeting of cry1Ac protein to the chloroplast using the rbcS:tp system confers a high level of plant protection to insects, thus providing an alternative strategy for crop insect management.

Anti-diabetic effects of compound K versus metformin versus compound K-metformin combination therapy in diabetic db/db mice.

Compound K (CK) is a major intestinal metabolite of ginsenosides derived from ginseng radix. In our preliminary studies, CK has shown to exhibit anti-hyperglycemic effect through its insulin-secreting action, similar to that of insulin secretagogue sulfonylureas. Metformin, a biguanide, improves insulin resistance by reducing gluconeogenesis and enhancing peripheral glucose uptake, promoting reduction of the plasma glucose level. The aim of this study was to compare the anti-diabetic effects of CK and metformin due to differences in their mechanisms of action and also to investigate whether treatment of CK and metformin in combination show synergistic or additive effects compared to each drug alone. Seven week-old male db/db mice were treated for 8 weeks. CK was given at a dose of 10 mg/kg, metformin at 150 mg/kg and the same dosage of each drug was applied to CK plus metformin combination group. Significant improvements were observed in plasma glucose and insulin levels, homeostasis model assessment-insulin resistance (HOMA-IR) index and in hematoxylin and eosin-stained liver tissues in combination group. Although further studies to elucidate the benefits of co-administration of CK and metformin are needed, our findings may provide basis to the discovery of a new combination therapy on diabetes control in type 2 diabetics.

In vivo and in vitro anti-inflammatory activities of alpha-linolenic acid isolated from Actinidia polygama fruits.

The fruit of Actinidia polygama (AP) has long been used as a folk medicine in Korea for the treatment of pain, rheumatoid arthritis and inflammation. In the present study, bioassay-guided fractionation of AP led to the separation and identification of a polyunsaturated fatty acid, alpha-linolenic acid (ALA), which was found to show anti-inflammatory activity. The anti-inflammatory effects of ALA, using acetic acid or carrageenan-induced inflammation models, were investi gated in mice or rats, respectively. ALA significantly inhibited the acetic acid-induced vascular permeability in a dose dependent manner (34.2 and 37.7% inhibition at doses of 5 and 10 mg/ kg, respectively). ALA also significantly reduced a rat paw edema induced by a single treatment of carrageenan. To investigate the mechanism of the anti-inflammatory action of ALA, the effects of ALA on lipopolysaccharide (LPS)-induced responses in the murine mac rophages cell line, RAW 264.7, were examined. Exposure of LPS-stimulated cells to ALA inhibited the accumulation of nitrite and prostaglandin E2 (PGE2) in the culture medium. Consistent with these observations, the protein and mRNA expression levels of iNOS and COX-2 enzyme were markedly inhibited by ALA in a dose dependent manner. These results suggest that the anti-inflammatory activity of ALA might be due to the suppression of the expressions of iNOS and COX-2 mRNA.

Role of nicotinic acetylcholine receptors in the regulation of kainic acid-induced hippocampal cell death in mice.

Kainic acid (KA) is a well-known excitatory, neurotoxic substance. In mice, morphological damage of hippocampus induced by KA administered intracerebroventricularly (i.c.v.) was markedly concentrated on the CA3 pyramidal neurons. In the present study, the possible role of nicotinic acetylcholine receptors (nAchRs) in hippocampal cell death induced by KA (0.1 microg) administered i.c.v. was examined. Methyllycaconitine (MC; nAchRs antagonist, 20 microg) attenuated KA-induced CA3 pyramidal cell death. KA increased immunoreactivities (IRs) of phorylated extracellular signal-regulated kinase (p-ERK; at 30 min), p-CaMK II (at 30 min), c-Fos (at 2 h), c-Jun (at 2 h), glial fibrillary acidic protein (GFAP at 1 day), and the complement receptor type 3 (OX-42; at 1 day) in hippocampal area. MC attenuated selectively KA-induced p-CaMK II, GFAP and OX-42 IR in the hippocampal CA3 region. Our results suggest that p-CaMK II may play as an important regulator responsible for the hippocampal cell death induced by KA administered i.c.v. in mice. Reactive astrocytes, which was meant by GFAP IR, and activated microglia, which was meant by OX-42 IR, may be a good indicator for measuring the cell death in hippocampal regions by KA-induced excitotoxicity. Furthermore, it is implicated that niconitic receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.

Antinociceptive profiles of platycodin D in the mouse.

Platycodin D (PD), one of several triterpene saponins, was isolated from roots of Platycodon grandiflorum. We previously reported that intracerebroventricular (i.c.v.) administration of PD showed an antinociceptive effect as measured by the tail-flick assay. However, its exact role in the regulation of antinociception in the various types of pain models has not yet been characterized. Thus, we attempted to find antinociceptive profiles of PD in various pain models. PD administered intraperitoneally (i.p.), i.c.v. or intrathecally (i.t.) showed antinociceptive effects in dose-dependent manners as measured by the tail-flick, writhing and formalin tests. In the tail-flick test, PD at the low doses reached the peak after 15 minutes and returned to the control level after 60 minutes. However, higher doses of PD showed a strong antinociception at least for 1 hour. PD administered i.t. showed stronger antinociception than that induced by i.c.v. administration PD in both tail-flick and writhing tests. In the formalin test, PD administered i.p., i.c.v. or i.t. showed antinociceptive effects during both the first (direct nociceptive stimulation) and second (late inflammatory) phases. Pretreatment with naltrexone i.p., i.c.v. or i.t. did not affect PD-induced inhibition of the tail-flick response. Our results suggest that PD shows a strong antinociceptive effect on the tail-flick, writhing and formalin tests, acting on central nervous system. However, PD-induced antinociception may not be mediated by the opioid receptors.

Regulation of c-fos and c-jun gene expression by lipopolysaccharide and cytokines in primary cultured astrocytes: effect of PKA and PKC pathways.

The effects of lipopolysaccharide (LPS) and several cytokines on the c-fos and c-jun mRNA expression were examined in primary cultured astrocytes. Either LPS (500 ng/mL) or interferon-gamma (IFN-gamma; 5 ng/mL) alone increased the level of c-fos mRNA (1 h). However, tumor necrosis factor-alpha (TNF-alpha; 10 ng/mL) or interleukin-1beta (IL-1beta; 5 ng/mL) alone showed no significant induction of the level of c-fos mRNA. TNF-alpha showed a potentiating effect in the regulation of LPS-induced c-fos mRNA expression, whereas LPS showed an inhibitory action against IFN-gamma-induced c-fos mRNA expression. LPS, but not TNF-alpha, IL-1beta and IFN-gamma, increased the level of c-jun mRNA (1 h). TNF-alpha and IFN-gamma showed an inhibitory action against LPS-induced c-jun mRNA expression. Both phorbol 12-myristate 13-acetate (PMA; 2.5 mM) and forskolin (FSK; 5 mM) increased the c-fos and c-jun mRNA expressions. In addition, the level of c-fos mRNA was expressed in an antagonistic manner when LPS was combined with PMA. When LPS was co-treated with either PMA or FSK, it showed an additive interaction for the induction of c-jun mRNA expression. Our results suggest that LPS and cytokines may be actively involved in the regulation of c-fos and c-jun mRNA expressions in primary cultured astrocytes. Moreover, both the PKA and PKC pathways may regulate the LPS-induced c-fos and c-jun mRNA expressions in different ways.

Roles of adenosine receptors in the regulation of kainic acid-induced neurotoxic responses in mice.

Kainic acid (KA) is a well-known excitatory and neurotoxic substance. In ICR mice, morphological damage of hippocampus induced by KA administered intracerebroventricularly (i.c.v.) was markedly concentrated on the hippocampal CA3 pyramidal neurons. In the present study, the possible role of adenosine receptors in hippocampal cell death induced by KA (0.1 microg) administered i.c.v. was examined. It has been shown that 3,7-dimethyl-1-propargylxanthine (DMPX; A2 adenosine receptors antagonist, 20 microg) reduced KA-induced CA3 pyramidal cell death. KA dramatically increased the phosphorylated extracellular signal-regulated kinase (p-ERK) immunoreactivities (IR) in dentate gyrus (DG) and mossy fibers. In addition, c-Jun, c-Fos, Fos-related antigen 1 (Fra-1) and Fos-related antigen 2 (Fra-2) protein levels were increased in hippocampal area in KA-injected mice. DMPX attenuated KA-induced p-ERK, c-Jun, Fra-1 and Fra-2 IR. However, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX; A1 adenosine receptor antagonist, 20 microg) did not affect KA-induced p-ERK, c-Jun, Fra-1 and Fra-2 IR. KA also increased the complement receptor type 3 (OX-42) IR in CA3 region of hippocampus. DMPX, but not PACPX, blocked KA-induced OX-42 IR. Our results suggest that p-ERK and c-Jun may function as important regulators responsible for the hippocampal cell death induced by KA administered i.c.v. in mice. Activated microglia, which was detected by OX-42 IR, may be related to phagocytosis of degenerated neuronal elements by KA excitotoxicity. Furthermore, it is implicated that A2, but not A1, adenosine receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.

Antinociceptive profiles of crude extract from roots of Angelica gigas NAKAI in various pain models.

To characterize the antinociceptive profiles of Angelica gigas NAKAI (ANG; Korean angelica), methanol extract from the dried roots of ANG was made and mice were administered orally at the various doses (from 0.25 to 3 g/kg). ANG produced the increased latencies of the tail-flick and hot-plate paw-licking responses in a dose-dependent manner. In acetic acid-induced writhing test, ANG dose-dependently decreased writhing numbers. Moreover, the cumulative response time of nociceptive behaviors induced by intraplantar formalin injection was reduced during both the 1st and the 2nd phases in a dose-dependent manner in ANG-treated mice. Furthermore, oral administration of ANG did not cause licking, scratching and biting responses induced by TNF-alpha (100 pg), IFN-gamma (100 pg) or IL-1beta (100 pg) injected intrathecally (i.t.), especially at higher dose (3 g/kg). Additionally, in ANG treated mice, the cumulative nociceptive response time for i.t. administration of substance P or capsaicin was dose-dependently diminished. Finally, nociceptive responses elicited by i.t. injection of glutamate (20 microg), N-methyl-D-aspartic acid (60 ng), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (13 ng) or kainic acid (12 ng) were decreased by oral administration of ANG. Our results suggest that ANG produces antinociception via acting on the central nervous system and shows antinociceptive profiles in various pain models, especially inflammatory pain.

Antinociceptive effects of methysergide in various pain models.

Several studies have demonstrated that the nonselective opioid receptor antagonist naloxone produces a paradoxical antinociception in the formalin test. The opioid system is related to the serotonergic system for producing antinociception at the spinal level. Here we also asked whether systemic (i.p.) and intrathecal (i.t.) administrations of a nonselective serotonergic antagonist, methysergide, might produce paradoxical antinociception similar to naloxone in the mouse formalin test. A diluted formalin solution was injected into the mouse plantar region of the hind paw and the duration of licking responses was measured at periods of 0-5 min (1st phase) and 20-40 min (2nd phase) after formalin injection. Methysergide administered i.p. and i.t. showed an attenuated licking duration only in the 2nd phase. The effect observed in the 2nd phase was reversed in the 5,7-dihydroxytriptamine, but not N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine pretreated group of mice, suggesting that descending serotonergic, but not noradrenergic, systems are involved in the methysergide antinociception. To further investigate the mechanism by which methysergide inhibited the nociceptive behaviors induced by formalin, the antinociceptive effect of methysergide was also tested in substance P (i.t.) and excitatory amino acids (i.t.), such as glutamate, N-methyl-D-aspartic acid, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainic acid, which are major components in the formalin-induced nociceptive transmission in the spinal cord pain models. The duration of nociceptive behaviors shown in these models was significantly shortened by i.p. and i.t. administration of methysergide. These results suggest that methysergide also produces a paradoxical antinociception in various pain models including the formalin test, similar to the results of naloxone.

Role of glutamate receptors and an on-going protein synthesis in the regulation of phosphorylation of Ca2+/calmodulin-dependent protein kinase II in the CA3 hippocampal region in mice administered with kainic acid intracerebroventricularly.

In an immunohistochemical study, kainic acid (KA, 0.1 microg) administered intracerebroventricularly (i.c.v.) dramatically increased the expression of Ca2+/calmodulin-dependent protein kinase II (CaMK II) and the phosphorylation of CaMK II (p-CaMK II) in the CA3 hippocampal region of mice. Pre-treatment with cycloheximide (a protein synthesis inhibitor; 200 mg/kg) intraperitoneally prevented the expression of CaMK II and phosphorylation of CaMK II induced by KA. In addition, pre-treatment with MK-801 ((5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine; an NMDA receptor blocker, 1 microg, i.c.v.) or CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; a non-NMDA receptor blocker, 0.5 microg, i.c.v.) attenuated the p-CaMK II, but not CaMK II, expression induced by KA. Our results suggest that KA administered supraspinally induces CaMK II and the phosphorylation of CaMK II expression in the CA3 hippocampal region, for which an on-going protein synthesis is needed. Furthermore, both NMDA and non-NMDA receptors appear to be involved in supraspinally administered KA-induced phosphorylation of CaMK II.

Possible antinociceptive mechanisms of opioid receptor antagonists in the mouse formalin test.

It has been reported that opioid receptor antagonist can induce antinociception in several nociceptive tests. In the intraplantar formalin pain model, however, opioid antagonist-induced antinociception, as well as its underlying mechanism, has not been well characterized. Therefore, in the mouse formalin test, we attempted to characterize the site of action and the possible opioid receptor subtypes. We found that naltrexone (a nonselective opioid antagonist) injected intraperitoneally (i.p., 1-20 mg/kg), intrathecally (i.t., 0.1-10 microg) and intracerebroventricularly (i.c.v., 0.1-10 microg) phase. Administration of beta-funaltrexamine (beta-FNA, 10-40 mg/kg i.p., 1.25-5 microg it or i.c.v.), naltrindole (1-10 mg/kg i.p., 1.25-5 microg it or i.c.v.) and nor-binaltorphimine (nor-BNI, 1-10 mg/kg i.p., 10-40 microg it or i.c.v.), which are selective mu-, delta- and kappa-opioid antagonists, respectively, also produced antinociception during the second phase. Additionally, we examined the involvement of the descending monoaminergic systems in the naltrexone-induced antinociception in the formalin test. Pretreatment with 5,7-dihydroxytryptamine (5,7-DHT, a serotonergic neurotoxin, 20 microg i.t.), but not N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4, a noradrenergic neurotoxin, 20 microg i.t.), reversed the naltrexone-induced antinociception during the second phase. Our results suggest that blockade of supraspinally or spinally located opioid receptors may play roles in the regulation of antinociception during the tonic painful stage. In addition, opioid receptors localized at the neuroterminal of the descending serotonergic, but not noradrenergic, inhibitory system in the spinal cord appear to be involved in opioid antagonist-induced antinociception during the second tonic phase of the formalin test.

Effect of ginsenoside Rd on nitric oxide system induced by lipopolysaccharide plus TNF-alpha in C6 rat glioma cells.

Effects of ginsenosides on nitric oxide (NO) production induced by lipopolysaccharide plus TNF-alpha (LNT) were examined in C6 rat glioma cells. Among several ginsenosides, ginsenoside Rd showed a complete inhibition against LNT-induced NO production. Ginsenoside Rd attenuated LNT-induced increased phosphorylation of ERK. Among several immediate early gene products, only Jun B and Fra-1 protein levels were increased by LNT, and ginsenoside Rd attenuated Jun B and Fra-1 protein levels induced by LNT. Furthermore, LNT increased AP-1 DNA binding activities, which were partially inhibited by ginsenoside Rd. Our results suggest that ginsenoside Rd exerts an inhibitory action against NO production via blocking phosphorylation of ERK, in turn, suppressing immediate early gene products such as Jun B and Fra-1 in C6 glioma cells.

Cycloheximide inhibits neurotoxic responses induced by kainic acid in mice.

In the present study, we examined the effect of cycloheximide on various pharmacological responses induced by kainic acid (KA) administered intracerebroventricularly (i.c.v.) in mice. In a passive avoidance test, a 20-min cycloheximide (200mg/kg, i.p.) pretreatment prevented the memory impairment induced by KA. The morphological damage induced by KA (0.1microg) in the hippocampus was markedly concentrated in the CA3 pyramidal neurons and cycloheximide effectively prevented the KA-induced pyramidal cell death in CA3 hippocampal region. In immunohistochemical study, KA dramatically increased the phosphorylation of extracellular signal-regulated protein kinase (p-ERK), c-Jun N-terminal kinase 1 (p-JNK1), and calcium/calmodulin-dependent protein kinase II (p-CaMK II). Cycloheximide attenuated the increased p-ERK, p-JNK1, and p-CaMK II levels induced by KA. Furthermore, cycloheximide inhibited the increased c-Fos and c-Jun protein expression levels induced by KA in the hippocampus. The activation of microglia was detected in KA-induced CA3 cell death region by immunostaining with a monoclonal antibody against the OX-42. Cycloheximide inhibited KA-induced increase of OX-42 immunoreactivity. Our results suggest that the increased expression of the c-Fos, c-Jun, and phosphorylation of ERK, JNK1, and CaMK II proteins may play important roles in the memory impairment and the cell death in CA3 region of the hippocampus induced by i.c.v. KA administration in mice. Furthermore, the activated microglia may be related to phagocytosis of degenerated neuronal elements induced by KA.

Isolation and characterization of the TRP1 gene from the yeast Yarrowia lipolytica and multiple gene disruption using a TRP blaster.

The TRP1 gene encoding N-(5'-phosphoribosyl)-anthranilate isomerase was isolated from the yeast Yarrowia lipolytica, in which only a few genetic marker genes are available. The Y. lipolytica TRP1 gene (YlTRP1) cloned by complementation of Y. lipolytica trp1 mutation was found to be a functional homologue of Saccharomyces cerevisiae TRP1. Since YlTRP1 could be used for counterselection in medium containing 5-fluoroanthranilic acid (5-FAA), we constructed TRP blasters that contained YlTRP1 flanked by a direct repeat of a sequence and allowed the recycling of the YlTRP1 marker. Using the TRP blasters the sequential disruption of target genes could be carried out within the same strain of Y. lipolytica. The nucleotide sequence of the YlTRP1 gene has been deposited at GenBank under Accession No. AF420590.

Antinociceptive mechanisms of orally administered decursinol in the mouse.

Antinociceptive profiles of decursinol were examined in ICR mice. Decursinol administered orally (from 5 to 200 mg/kg) showed an antinociceptive effect in a dose-dependent manner as measured by the tail-flick and hot-plate tests. In addition, decursinol attenuated dose-dependently the writhing numbers in the acetic acid-induced writhing test. Moreover, the cumulative response time of nociceptive behaviors induced by an intraplantar formalin injection was reduced by decursinol treatment during the both 1st and 2nd phases in a dose-dependent manner. Furthermore, the cumulative nociceptive response time for intrathecal (i.t.) injection of TNF-alpha (100 pg), IL-1 beta (100 pg), IFN-gamma (100 pg), substance P (0.7 microg) or glutamate (20 microg) was dose-dependently diminished by decursinol. Intraperitoneal (i.p.) pretreatment with yohimbine, methysergide, cyproheptadine, ranitidine, or 3,7-dimethyl-1-propargylxanthine (DMPX) attenuated inhibition of the tail-flick response induced by decursinol. However, naloxone, thioperamide, or 1,3-dipropyl-8-(2-amino-4-chloro-phenyl)-xanthine (PACPX) did not affect inhibition of the tail-flick response induced by decursinol. Our results suggests that decursinol shows an antinociceptive property in various pain models. Furthermore, antinociception of decursinol may be mediated by noradrenergic, serotonergic, adenosine A(2), histamine H(1) and H(2) receptors.