Adiponectin stimulates glucose uptake in mouse blastocysts and embryonic carcinoma cells.

Preimplantation embryos are sensitive to maternal hormones affecting embryonic signal transduction and metabolic functions. We examined whether adiponectin, the most abundantly secreted adipokine, can influence glucose transport in mouse embryonic cells. In mouse blastocysts full-length adiponectin stimulated glucose uptake, while no effect of globular adiponectin was found. Full-length adiponectin stimulated translocation of GLUT8 glucose transporter to the cell membrane; we did not detect significant changes in the intracellular localization of GLUT4 glucose transporter in adiponectin-treated blastocysts. To study adiponectin signaling in detail, we used embryoid bodies formed from mouse embryonic carcinoma cell (ECC) line P19. We confirmed the expression of adiponectin receptors in these cells. Similar to mouse blastocysts, full-length adiponectin, but not globular adiponectin, stimulated glucose uptake in ECC P19 embryoid bodies. Moreover, full-length adiponectin stimulated AMPK and p38 MAPK phosphorylation. These results indicate that besides AMPK, p38 MAPK is a potential target of adiponectin in mouse embryonic cells. AMPK inhibitor did not influence the adiponectin-stimulated p38 MAPK phosphorylation, indicating independent action of these two signaling pathways. In mouse embryos adiponectin acts as a hormonal regulator of glucose uptake, which becomes especially important in phases with reduced levels of circulating insulin. Our results suggest that adiponectin maintains the glucose supply for early embryos under hypoinsulinaemic conditions, for example, in mothers suffering from type 1 diabetes mellitus.

The laboratory performance of the enFLOW(®) , buddy lite(™) and ThermoSens(®) fluid warmers.

We measured heating of isotonic saline by three fluid warmers in six experiments: saline at 5 °C or 20 °C delivered at 30, 50 or 100 ml.min(-1) . At the three flow rates, the enFLOW(®) , buddy lite(™) and ThermoSens(®) systems heated 5 °C saline to mean (SD) temperatures of: 41.1 (0.5) °C, 37.7 (0.6) °C and 39.1 (0.6) °C; to 40.3 (0.8) °C, 33.9 (1.6) °C and 39.3 (0.7) °C; and to 37.1 (0.8) °C, 24.0 (1.3) °C and 37.6 (1.0) °C, respectively, p < 0.0001 for each experiment. The mean (SD) times taken to heat 5 °C saline were: 16.6 (1.7) s, 258.4 (58.9) s and 134.2 (79.6) s; 16.9 (1.8) s, 256.2 (62.2) s and 182.5 (74.5) s; and 21.5 (1.5) s, 275.9 (49.3) s and 313.5 (18.0) s, respectively, p < 0.0003 for each experiment. The results for saline at 20 °C were similar. The enFLOW system heated saline above 36 °C faster than the ThermoSens system, whereas the buddy lite often failed to achieve 36 °C.

Rapid detection of antibiotic resistance based on mass spectrometry and stable isotopes.

With the emergence and growing complexity of bacterial drug resistance, rapid and reliable susceptibility testing has become a topical issue. Therefore, new technologies that assist in predicting the effectiveness of empiric antibiotic therapy are of great interest. Although the use of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid detection of antibiotic resistance is an attractive option, the current methods for MALDI-TOF MS susceptibility testing are restricted to very limited conditions. Here, we describe a technique that may allow for rapid susceptibility testing to an extent that is comparable to phenotypic methods. The test was based on a stable isotope labelling by amino acids in cell culture (SILAC)-like approach. This technique was used to visualise the growth of bacteria in the presence of an antibiotic. Pseudomonas aeruginosa was chosen as the model organism, and strains were incubated in normal medium, medium supplemented with (13)C6-(15) N2-labelled lysine and medium supplemented with labelled lysine and antibiotic. Peak shifts occurring due to the incorporation of the labelled amino acids were detected by MALDI-TOF MS. Three antibiotics with different mechanisms of action, meropenem, tobramycin and ciprofloxacin, were tested. A semi-automated algorithm was created to enable rapid and unbiased data evaluation. With the proposed test, a clear distinction between resistant and susceptible isolates was possible for all three antibiotics. The application of SILAC technology for the detection of antibiotic resistance may contribute to accelerated and reliable susceptibility testing.

Neutron Laue diffraction study on the magnetic phase diagram of multiferroic MnWO4 under pulsed high magnetic fields.

We have combined time-of-flight neutron Laue diffraction and pulsed high magnetic fields at the Spallation Neutron Source to study the phase diagram of the multiferroic material MnWO(4). The control of the field-pulse timing enabled an exploration of magnetic Bragg scattering through the time dependence of both the neutron wavelength and the pulsed magnetic field. This allowed us to observe several magnetic Bragg peaks in different field-induced phases of MnWO(4) with a single instrument configuration. These phases were not previously amenable to neutron diffraction studies due to the large fields involved.

Time-resolved measurements of PM2.5 carbonaceous aerosols at Gosan, Korea.

In order to better understand the characteristics of atmospheric carbonaceous aerosol at a background site in Northeast Asia, semicontinuous organic carbon (OC) and elemental carbon (EC), and time-resolved water-soluble organic carbon (WSOC) were measured by a Sunset OC/ EC and a PILS-TOC (particle-into-liquid sampler coupled with an online total organic carbon) analyzer, respectively, at the Gosan supersite on Jeju Island, Korea, in the summer (May 28-June 17) and fall (August 24-September 30) of 2009. Hourly average OC concentration varied in the range of approximately 0.87-28.38 microgC m-3, with a mean of 4.07+/- 2.60 microgC m-3, while the hourly average EC concentration ranged approximately from 0.04 to 8.19 .microgC m-3, with a mean of 1.35 +/- 0.71 microgC m-3, from May 28 to June 17, 2009. During the fall season, OC varied in the approximate range 0.9-9.6 microgC m-3, with a mean of 2.30 +/-0.80 microgC m-3, whereas EC ranged approximately from 0.01 to 5.40 microgC m-3, with a mean of 0.66 +/- 0.38 microgC m-3. Average contributions of EC to TC and WSOC to OC were 26.0% +/- 9.7% and 20.6% +/-7.4%, and 37.6% +/- 23.5% and 57.2% +/- 22.2% during summer and fall seasons, respectively. As expected, clear diurnal variation of WSOC/OC was found in summer, varying from 0.22 during the nighttime up to 0.72 during the daytime, mainly due to the photo-oxidation process. In order to investigate the effect of air mass pathway on the characteristics of carbonaceous aerosol, 5-day back-trajectory analysis was conducted using the HYSPLIT model. The air mass pathways were classified into four types: Continental (CC), Marine (M), East Sea (ES) and Korean Peninsula (KP). The highest OC/EC ratio of 3.63 was observed when air mass originated from the Continental area (CC). The lowest OC/EC ratio of 0.79 was measured when air mass originated from the Marine area (M). A high OC concentration was occasionally observed at Gosan due to local biomass burning activities. The contribution of secondary OC to total OC varied approximately between 8.4% and 32.2% and depended on air mass type.

Characterization of the hypothalamic proopiomelanocortin gene and beta-endorphin expression in the hypothalamic arcuate nucleus of mice elicited by inflammatory pain.

We examined proopiomelanocortin (POMC) mRNA and beta-endorphin expression in the hypothalamus of mice after various nociceptive stimuli. The time-course study (10 min, 30 min, 1 h, 2 h, and 10 h) showed that the POMC mRNA level significantly increases from 1 h after s.c. formalin injection and returns to the control level at 10 h. Intrathecal (i.t.) substance P (SP) injection also increases the hypothalamic POMC mRNA level from 1 h to 10 h. However, i.t. glutamate injection did not affect the hypothalamic POMC gene expression at all time points. We found that the POMC mRNA after s.c. formalin injection was located in the arcuate nucleus of the hypothalamus. In the same manner, beta-endorphin immunoreactivity was also increased in the hypothalamic arcuate nucleus. The expression of phosphorylated extracellular signal-regulated protein kinase 1/2 (pERK1/2), phosphorylated calcium/calmodulin-dependent protein kinase-IIalpha (pCaMK-IIalpha) protein and phosphorylated IkappaB (pIkappaB) protein was increased by s.c. formalin injection at various time points. We also found that increased pERK1/2, pCaMKIIalpha and pIkappaB protein after s.c. formalin injection was mainly located in the arcuate nucleus of hypothalamus in which cells containing beta-endorphin after s.c. formalin injection also express pERK1/2, pCaMK-IIalpha and pIkappaB immunoreactivity. In addition, formalin-induced POMC mRNA expression was significantly reduced by 10 min, pretreatment with i.c.v. PD98059 (mitogen-activated protein kinase (MAPK) pathways inhibitor; 6.6 mug) and KN93 (pCaMK-II inhibitor; 20 mug). In conclusion, POMC mRNA expression in the arcuate nucleus of the hypothalamus was increased by inflammatory pain stimuli, in which pERK1/2, pCaMK-IIalpha and NFkappaB may play an important role in the expression of the hypothalamic POMC gene and beta-endorphin expression.

Differential expression of phosphorylated Ca2+/calmodulin-dependent protein kinase II and phosphorylated extracellular signal-regulated protein in the mouse hippocampus induced by various nociceptive stimuli.

In the present study, we characterized differential expressions of phosphorylated Ca(2+)/calmodulin-dependent protein kinase IIalpha (pCaMKIIalpha) and phosphorylated extracellular signal-regulated protein (pERK) in the mouse hippocampus induced by various nociceptive stimuli. In an immunoblot study, s.c. injection of formalin and intrathecal (i.t.) injections of glutamate, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1 beta) significantly increased pCaMKIIalpha expression in the hippocampus, but i.p. injections of acetic acid did not. pERK1/2 expression was also increased by i.t. injection of glutamate, TNF-alpha, and IL-1beta but not by s.c. injections of formalin or i.p. injections of acetic acid. In an immunohistochemical study, we found that increased pCaMKIIalpha and pERK expressions were mainly located at CA3 or the dentate gyrus of the hippocampus. In a behavioral study, we assessed the effects of PD98059 (a MEK 1/2 inhibitor) and KN-93 (a CaMKII inhibitor) following i.c.v. administration on the nociceptive behaviors induced by i.t. injections of glutamate, pro-inflammatory cytokines (TNF-alpha or IL-1beta), and i.p. injections of acetic acid. PD98059 as well as KN-93 significantly attenuated the nociceptive behavior induced by glutamate, pro-inflammatory cytokines, and acetic acid. Our results suggest that (1) pERKalpha and pCaMK-II located in the hippocampus are important regulators during the nociceptive processes induced by s.c. formalin, i.t. glutamate, i.t. pro-inflammatory cytokines, and i.p. acetic acid injection, respectively, and (2) the alteration of pERK and pCaMKIIalpha in nociceptive processing induced by formalin, glutamate, pro-inflammatory cytokines and acetic acid was modulated in a different manner.

The effect of formalin pretreatment on nicotine-induced antinociceptive effect: the role of mu-opioid receptor in the hippocampus.

Nicotine is attractive as an analgesic component despite that its antinociceptive mechanism is not well known until now. In the present study, we examined the antinociceptive effect of nicotine administered supra-spinally on acetic acid-induced visceral pain induction (writhing test), and found that the antinociceptive effect of nicotine was abolished by mu-, delta-, and kappa-opioid receptor antagonist administered i.c.v. In addition, s.c. 5% formalin pretreatment at 5 h, 20 h, 40 h, and 1 week prior to i.c.v. nicotine injection abolished the antinociceptive effect of nicotine in the writhing test, suggesting that s.c. formalin pretreatment induced tolerance to the antinociceptive effect of nicotine in the supra-spinal region. Furthermore, neuronal loss of the hippocampal cornus ammonis (CA) 3 region reduced nicotine-induced an antinociceptive effect in the writhing test. In Western blot assay, we examined s.c. formalin injection down-regulated mu-opioid receptor in the hippocampus after 40 h, and its effect was maintained for 1 week. However, various acetylcholine receptor subunits and delta-, and kappa-opioid receptors were not altered. These results suggest that s.c. formalin pretreatment can contribute to induce tolerance on nicotine-induced antinociception as down-regulating mu-opioid receptor in the hippocampus, especially 40 h after s.c. formalin injection.

Multicenter study of intravenous busulfan, cyclophosphamide, and etoposide (i.v. Bu/Cy/E) as conditioning regimen for autologous stem cell transplantation in patients with non-Hodgkin's lymphoma.

The current study aimed to evaluate the efficacy and toxicity of a combination of intravenous busulfan, cyclophosphamide and etoposide (i.v. Bu/Cy/E) as a conditioning regimen prior to autologous hematopoietic stem cell transplantation in patients with non-Hodgkin's lymphoma (NHL). Sixty-four patients with relapsed/refractory (n=36) or high-risk (n=28) lymphoma were enrolled. The high-dose chemotherapy consisted of i.v. Bu (0.8 mg kg(-1) i.v. q 6 h from day -7 to day -5), Cy (50 mg kg(-1) i.v. on day -3 and day -2) and E (400 mg m(-2) i.v. on day -5 and day -4). The median age was 43 (range 18-65) years, and 39 patients were male. Diffuse large B-cell lymphoma (40.6%) was the most common histological subtype. All evaluable patients achieved an engraftment of neutrophils (median, day 12) and platelets (median, day 13). Hepatic veno-occlusive disease was observed in four patients (three mild, one moderate grade), and two patients (3.1%) died from treatment-related complications. At a median follow-up of 16.4 months, 15 patients (23.4%) exhibited a relapse or progression, while 13 patients (20.3%) had died of disease. The estimated 3-year overall and progression-free survival for all patients was 72.1 and 70.1%, respectively. In conclusion, the conditioning regimen of i.v. Bu/Cy/E was well tolerated and seemed to be effective in patients with aggressive NHL.

Unique structural features of a BCL-2 family protein CED-9 and biophysical characterization of CED-9/EGL-1 interactions.

The interactions between B-cell lymphoma 2 (BCL-2) family members are known to be mediated through the binding of the BH3 domain of a proapoptotic member to the BH3-binding groove of an antiapoptotic member. We determined the crystal structure of antiapoptotic CED-9, which reveals a unique C-terminal helix altering the common BH3-binding region. A coexpression system to produce CED-9 in complex with proapoptotic EGL-1 enabled us to show that the binding of EGL-1 to CED-9 is extremely stable, raising the melting temperature (T(M)) of CED-9 by 25 degrees C, and that the binding surface of CED-9 extends beyond the BH3-binding region and reaches the BH4 domain. Consistently, the T(M) and a 1H-15N correlation NMR spectrum of CED-9 in complex with EGL-1 are drastically different from those of CED-9 in complex with the EGL-1 BH3 peptide. The data suggest that the recognition between other BCL-2 family members may also involve much wider protein surfaces than is previously thought.

Central injection of nitric oxide synthase inhibitors increases peripheral interleukin-6 and serum amyloid A: involvement of adrenaline from adrenal medulla.

1. Accumulating evidence suggests that plasma levels of interleukin-6 (IL-6), a major cytokine stimulating the synthesis of acute phase proteins, are intimately regulated by the central nervous system (CNS). 2. In the present study, effects of intracerebroventricular (i.c. v) injection of N(G)-nitro-L-arginine methyl ester (L-NAME) or 7-nitroindazole, nitric oxide synthase (NOS) inhibitors, on plasma IL-6 levels and peripheral IL-6 mRNA expression were examined in mice. 3. L-NAME (0.1 - 2 microg per mouse i.c.v.) and 7-nitroindazole (0.2 - 2 microg per mouse i.c.v.) induced a dose-dependent increase in plasma IL-6 levels and a subsequent increase in circulating serum amyloid A, a liver acute-phase protein. In contrast, an intraperitoneal (i.p.) injection of L-NAME up to the dose of 25 microg per mouse had no effect. 4. Pretreatment with yohimbine (alpha(2)-adrenergic antagonist; 1 mg kg(-1) i.p.), or ICI-118,551 (beta(2)-adrenergic antagonist; 2 mg kg(-1) i.p.), but not with prazosin (alpha(1)-adrenergic antagonist; 1 mg kg(-1) i.p.), nor betaxolol (beta(1)-adrenergic antagonist; 2 mg kg(-1) i.p.), significantly inhibited the central L-NAME-induced plasma IL-6 levels. 5. I.c.v. (50 microg per mouse) or i.p. (100 mg kg(-1)) pretreatment with 6-hydroxydopamine had no effect on central L-NAME-induced plasma IL-6 levels. However, intrathecal (i.t.) pretreatment with 6-hydroxydopamine (20 microg per mouse) markedly inhibited central L-NAME-induced plasma IL-6 levels. Both yohimbine (1.5 microg per mouse i.t.) and ICI-118,551 (1.5 microg per mouse i. t.) were effective in inhibition of central L-NAME-induced plasma IL-6 levels. 6. There was an elevation of base-line plasma IL-6 levels in adrenalectomized animals. The adrenalectomy-enhanced levels were not further increased by central L-NAME. 7. L-NAME (2 microg per mouse i.c.v.) induced an increase in IL-6 mRNA expression in liver, spleen, and lymph node. 8. These results suggest that NOS activity in the brain tonically down-regulates peripheral IL-6 by inhibiting adrenaline release from the adrenal medulla.

Protection against beta-amyloid peptide toxicity in vivo with long-term administration of ferulic acid.

1. beta-Amyloid peptide (A beta), a 39 -- 43 amino acid peptide, is believed to induce oxidative stress and inflammation in the brain, which are postulated to play important roles in the pathogenesis of Alzheimer's disease. Ferulic acid is an antioxidant and anti-inflammatory agent derived from plants; therefore, the potential protective activity of ferulic acid against A beta toxicity in vivo was examined. 2. Mice were allowed free access to drinking water (control) or water containing ferulic acid (0.006%). After 4 weeks, A beta 1-42 (410 pmol) was administered via intracerebroventricular injection. 3. Injection of control mice with A beta 1-42 impaired performance on the passive avoidance test (35% decrease in step-through latency), the Y-maze test (19% decrease in alternation behaviour), and the water maze test (32% decrease in percentage time in platform-quadrant). In contrast, mice treated with ferulic acid prior to A beta 1-42 administration were protected from these changes (9% decrease in step-through latency; no decrease in alternation behaviour; 14% decrease in percentage time in platform-quadrant). A beta 1-42 induced 31% decrease in acetylcholine level in the cortex, which was tended to be ameliorated by ferulic acid. 4. In addition, A beta 1-42 increased immunoreactivities of the astrocyte marker glial fibrillary acidic protein (GFAP) and interleukin-1 beta (IL-1 beta) in the hippocampus, effects also suppressed by pretreatment with ferulic acid. 5. Administration of ferulic acid per se unexpectedly induced a transient and slight increase in GFAP and IL-1 beta immunoreactivity in the hippocampus on day 14, which returned to basal levels on day 28. A slight (8%) decrease in alternation behaviour was observed on day 14. 6. These results demonstrate that long-term administration of ferulic acid induces resistance to A beta 1-42 toxicity in the brain, and suggest that ferulic acid may be a useful chemopreventive agent against Alzheimer's disease.

Difference in H2O2 toxicity between intact renal tubules and cultured proximal tubular cells.

The present study was undertaken to examine the response to H2O2 and t-butylhydroperoxide (t-BHP) in various in vitro model systems of renal proximal tubules: rabbit renal cortical slices, freshly isolated rabbit proximal tubules, rabbit primary cultured proximal tubular cells, and opossum kidney (OK) cells. t-BHP increased lactate dehydrogenase release and lipid peroxidation in a concentration-dependent manner over the concentration range of 0.2 to 3 mM in cortical slices, whereas H2O2 caused a similar concentration-dependent increase in both parameters at 5-100 mM. The sensitivity of isolated tubules to both peroxides was similar to that of cortical slices. In primary cultured cells and OK cells, however, the cytotoxicity of H2O2 was identical to that of t-BHP. The cytotoxicity of t-BHP was not different among all the systems examined. The specific activity of catalase in cortical slices was similar to that of isolated tubules, but it was much higher than that of primary cultured cells or opossum kidney cells. Glutathione (GSH) peroxidase activity was not different among all the systems examined. The expression of catalase mRNA in cortical slices and isolated tubules was higher than that in primary cultured cells, whereas those of superoxide dismutase, glutathione peroxidase, or beta-actin were not different among the systems. These results indicate that intact proximal tubules are more resistant to H2O2 than are cultured proximal tubular cells, and the resistance is due to a higher specific activity of catalase resulting from the increased expression of its mRNA.

Cisplatin-induced apoptosis by translocation of endogenous Bax in mouse collecting duct cells.

cis-platinum(II) (cis-diammine dichloroplatinum; cisplatin) is a potent antitumor compound that is widely used for the treatment of many malignancies. An important side-effect of cisplatin is nephrotoxicity, which results from injury to renal tubular epithelial cells and can be manifested as either acute renal failure or a chronic syndrome characterized by renal electrolyte wasting. Recently, apoptosis has been recognized as an important mechanism of cell death mediating the antitumor effect of cisplatin. This study was undertaken to examine the mechanisms of cell death induced by cisplatin in M-1 cells, which were derived from the outer cortical collecting duct cells of SV40 transgenic mice. Treatment of M-1 cells with high concentrations of cisplatin (0.5 and 1 mM) for 2 hr led to necrotic cell death, whereas a 24-hr treatment with 5-20 microM cisplatin led to apoptosis. Antioxidants protected against cisplatin-induced necrosis, but not apoptosis, indicating that reactive oxygen species play a role in mediating necrosis but not apoptosis induced by cisplatin and that the mechanism of cell death induced by cisplatin is concentration dependent. The low concentrations of cisplatin, which induced apoptosis in M-1 cells, did not affect the expression levels of Bcl-2-related proteins and did not activate c-Jun NH2-terminal kinase (SAPK/JNK). Cisplatin induced the translocation of endogenous Bax from the cytosolic to the membrane fractions and, subsequently, the release of cytochrome c. Overexpression of Bcl-2 blocked cisplatin-induced apoptosis and Bax translocation. These observations suggest that the subcellular redistribution of Bax is a critical event in the apoptosis induced by cisplatin.

Detection of genetic alterations in the human gastric cancer cell lines by two-dimensional analysis of genomic DNA.

We examined genomic DNA from each of three human-derived gastric cancer cell lines, using the technique of restriction landmark genomic scanning (RLGS) which allows monitoring of approximately 2, 000 NotI landmarks. The resulting DNA spots from cancer cell DNA were compared with those in normal mucosa or gastric primary tumor. In all, 9 intense spots were detected from two of the three cancer cell lines. Two highly intensified spots were common in the two cancer cell lines and proven to be originated from DNA region containing the human c-myc proto-oncogene on chromosome 8. The degree of amplification of c-myc DNA was similar to each other and was estimated to be 60-fold as compared to those from normal mucosa DNA. On the basis of chromosome-assigned RLGS (CA-RLGS), other spots were assigned to each chromosome, such as one on chromosome 8, two each from chromosome 20, and three on one of chromosome 9-12. The remaining spot seems to be due to demethylation of a repetitive element. Twenty-four spot that were lost due to either homozygous deletion or methylation on corresponding NotI cleavage sites were commonly observed in all cancer cells. These spots were also assigned to each chromosome: one each from chromosome 2, 6, 7, 13, 14, 16, and 20, two each from chromosome 3 and 5, and nine from chromosome 9-12 by CA-RLGS. Many of the multi-copy spots corresponding to ribosomal RNA genes were greatly decreased due mainly to methylation on CpG islands along with minor rDNA variants, indicating that only minor rRNA genes may be silenced in these cancer cells. These results show that the present alterations detected by RLGS might be useful for identification of candidate genes inactivated or expressed unexpectedly in tumor development and tumor progression in the stomach.

Dexamethasone enhances phospholipase D activity in M-1 cells.

Phospholipase D (PLD) is an enzyme involved in signal transduction and widely distributed in mammalian cells. The signal transduction pathways and role for phospholipid metabolism during hormonal response in cortical collecting duct remain partly undefined. It has been reported that dexamethasone increases transepithelial transport in M-1 cells that are derived from the mouse cortical collecting duct. We investigated the expression and activity of PLD in M-1 cells. Basal PLD activity of M-1 cells cultured in the presence of dexamethasone (5 microM) was higher than in the absence of dexamethasone. Dexamethasone and ATP activated PLD in M-1 cells but phorbol ester did not stimulate PLD activity. Vasopressin, bradykinin, dibutyryl cyclic AMP, and ionomycin were ineffective in activating PLD of the cells. The PLD2 isotype was detected by immunoprecipitation but PLD1 was not detected in M-1 cells. Addition of GTPgammaS and ADP-ribosylation factor or phosphatidylinositiol 4,5-bisphosphate to digitonin-permeabilized cells did not augment PLD activity. In intact cells PLD activity was increased by sodium oleate but there was no significant change between dexamethasone treated- and untreated cells by oleate. These results suggest that at least two types of PLD are present in M-1 cells and PLD plays a role in the corticosteroid-mediated response of cortical collecting duct cells.

Inhibition of stress-induced plasma corticosterone levels by ginsenosides in mice: involvement of nitric oxide.

Ginseng total saponins (GTS) injected intracerebroventricularly (i.c.v.) at doses of 0.1-1 microg inhibited the i.c.v. injection stress-induced plasma corticosterone levels in mice. The inhibitory action of GTS was blocked by co-administered N(G)-nitro-L-arginine methyl ester (L-NAME; 1.5 microg, i.c.v.), an inhibitor of nitric oxide synthase (NOS). Of the ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, 20(S)-Rg3 and 20(R)-Rg3 injected i.c.v. at doses of 0.01-1 microg, 20(S)-Rg3 and Rc significantly inhibited the i.c.v. injection stress-induced plasma corticosterone levels. The inhibitory actions of 20(S)-Rg3 and Rc were blocked by co-administered L-NAME (1.5 microg, i.c.v.). These results suggest that GTS, 20(S)-Rg3 and Rc may inhibit the i.c.v. injection stress-induced hypothalamo-pituitary-adrenal response by inducing NO production in the brain.

Increased expression of the gentamicin resistance gene by a Tn3 sequence located at the upstream region.

Recombinant plasmids designated as pCH1 and pCH21 were constructed to determine the influence of the bla sequence on the expression of the gentamicin resistance gene which is located downstream of Tn3. Minimal inhibitory concentration (MIC) test and Northern hybridization results with the constructed plasmids showed that the transcription of the gentamicin resistance gene was initiated from the promoter, located downstream from the PstI site within the bla gene, and the hybrid promoter created by IR sequences of Tn3. Although the read-through transcription from the bla promoter did not occur, the transcription of the bla gene increased the expression of the gentamicin resistance gene.

Involvement of NMDA receptor in the regulation of plasma interleukin-6 levels in mice.

MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclopepten-5,10-imine maleate), a non-competitive NMDA receptor antagonist (0.01-1 micrograms), injected intracerebroventricularly (i.c.v.) dose dependently increased the baseline levels of plasma interleukin-6 in mice. In the 1-h immobilization-stressed animals, MK-801 (1 micrograms) administered i.c.v. produced an additive increase of plasma interleukin-6. NMDA (N-methyl-D-aspartate) (3, 10 ng) administered i.c.v. attenuated dose dependently the 1-h immobilization stress-induced rise in plasma interleukin-6 level. Neither 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) (0.01-0.5 micrograms) nor alpha-methyl-4-carboxyphenylglycine (MCPG) (1-20 micrograms), antagonists of non-NMDA and metabotropic glutamate receptors, respectively, i.c.v. administered, affected the basal and stress-induced plasma interleukin-6 levels. These data indicate that NMDA receptors may be involved in the suppressive regulation of the plasma interleukin-6 levels.

Increased plasma corticosterone, aggressiveness and brain monoamine changes induced by central injection of pertussis toxin.

The effects of intracerebroventricular (i.c.v.) injection of pertussis toxin, a specific inhibitor of G(i)/G(o) proteins, on plasma corticosterone levels, aggressiveness, and hypothalamic and hippocampal monoamines and their metabolites levels were examined in mice. Plasma corticosterone level was markedly increased at 3 h after pertussis toxin injection (0.03 and 0.2 microg/mouse), peaked at 6 h and was still increased for up to 6 days after injection. Mice injected with pertussis toxin (0.2 microg/mouse) did not show weight gain between day 0 and day 6 after injection. In addition, pertussis toxin (0.2 microg/mouse) induced a progressive increase in aggressiveness, i.e. a decrease in attack latency and an increase in number of attacks, on day 1 and 6 after injection. Brain monoamines and their metabolites levels were changed on day 1 and 6 after pertussis toxin injection (0.2 microg/mouse): in the hypothalamus, levels of dopamine and 3,4-dihydroxyphenylacetic acid were increased, norepinephrine level decreased, and 5-hydroxyindole acetic acid (5-HIAA) level was markedly increased, with no changes in 5-hydroxytryptamine (5-HT) level, whereas in the hippocampus, 5-HT level was significantly decreased, with no changes in 5-HIAA and catecholamines. These results suggest that signal transduction through G(i)/G(o) proteins in the brain is involved in the modulation of hypothalamo-pituitary-adrenal axis, aggressiveness, and monoamine levels in vivo.