Development of natural products as drugs acting on central nervous system

We have recenty studied several natural product constituents which have effects on the CNS. (1) Tetrahydropalmatine (THP) and its analogues were isolated from Corydalis ambigua and various species of Stephania. (+)-THP and (-)-THP posses not only analgesic activity, but also exert sedative-tranquillizing and hypnotic actions. Results of receptor binding assay and their pre-and post-synaptic effects on dopaminergic system indicate that (-)-THP and (-)-stepholidine are dopamine receptor antagonists while (+)-THP is a selective dopamine depletor. (2) 3-Acetylaconitine (AAC) is an alkaloid isolated from Aconitum flavum. The relative potency of analgesic action of AAC was 5.1-35.6 and 1250-3912 times that of morphine and aspirin, respectively. The analgesic effect of AAC was antagonized by naloxone, but was eliminated by reserpine. In monkeys, after AAC was injected for 92 days, no abstinence syndrome was seen after sudden AAC withdrawal or when challenged with nalorphine. (3) Huperzine A (Hup-A) is an alkaloid isolated from Huperzia serrata which was found to be a selective ChE inhibitor and could improve learning and retrieval process. Preliminary clinical studies showed that Hup-A improve short-and long-term memory in patients of cerebral arteriosclerosis with memory impairment. (4) Ranamargarin is a new tetradecapeptide isolated from the skin of the Chines frog Rana margaratae. This peptide may mainly act on NK-1 receptor

Modulating effect of adenosine deaminase on function of adenosine A 1 receptors 1

Aim : To study the modulating effect of adenosine deaminase (ADA) on yhe adenosine A 1 receptor (A 1 R) in HEK293 cells stably expressing the human A 1 R. Methods : cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned A 1 R cDNA was sequenced and stably expressed in HEK293 cells. The modulating effect of adenosine deaminase on A 1 R was studied by using [ 3 H]DPCPX binding assay and an intracellular calcium assay. Results : HEK293 cells stably expressing human A 1 R were obtained. Saturation studies showed that the K D value and B max value of [ 3 H]DPCPX were 1.6±0.2 nmol/L and 1.819±0.215 nmol/g of protein respectively, in the absence of ecto-ADA respectively, and 1.3±0.2 nmol/L and 1.992±0.130 nmol/g of protein in the presence of ecto-ADA respectively, suggesting that the K D value and B max value of [ 3 H]DPCPX were unaffected by ecto-ADA. In the case of [ 3 H]DPCPX competition curves obtained from intact cells or membranes, A 1 R agonist CCPA/[ 3 H]DPCPX competition curve could be fitted well to a one-site model in the absence of ecto-ADA and a two-site model in the presence of ecto-ADA with a K H value of 0.74 (0.11–4.8) nmol/L (intact cells) or 1.8 (0.25–10) nmol/L (membrane) and a K L value of 0.94 (0.62–1.41) Μmol/L (intact cells) or 0.77 (0.29–0.99) Μmol/L (membrane). The K L value is not significantly different from the EC 50 value of 0.84(0.57–1.23) Μmol/L (intact cells) or 0.84 (0.63–1.12) Μmol/L (membrane) obtained in the absence of ecto-ADA. Similar results were obtained from the CPA/[ 3 H]DPCPX competition curve in the absence or presence of ecto-ADA on intact cells or membranes. Intracellular calcium assay demonstrated that the EC 50 value of CPA were 10 (5–29) nmol/L and 94 (38–229) nmol/L in the presence or absence of ecto-ADA, respectively. Conclusion : A 1 R stably expressed in the HEK293 cells display a low affinity for agonists in the absence of ADA and high and low affinities for agonists in the presence of ADA. The presence of ADA may promote the signaling through the adenosine A 1 receptor in HEK293 cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73693/1/j.1745-7254.2005.00524.x.pd

Cloning, expression, and functional analysis of human dopamine D1 receptors

Aim : To construct an HEK293 cell line stably expressing human dopamine D 1 receptor (D 1 R). Methods : cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned D 1 R cDNA was sequenced and stably expressed in HEK293 cells. Expression of D 1 R in HEK293 cells was monitored by the [ 3 H]SCH23390 binding assay. The function of D 1 R was studied by the cAMP accumulation assay, CRE-SEAP reporter gene activity assay, and intracellular calcium assay. Results : An HEK293 cell line stably expressing human D 1 R was obtained. A saturation radioligand binding experiment with [ 3 H]SCH23390 demonstrated that the K d and B max values were 1.5±0.2 nmol/L and 2.94±0.15 nmol/g of protein, respectively. In the [ 3 H]SCH23390 competition assay, D 1 R agonist SKF38393 displaced [ 3 H]SCH23390 with an IC 50 value of 2.0 (1.5–2.8) Μmol/L. SKF38393 increased the intracellular cAMP level and CRE-SEAP activity through D 1 R expressed in HEK293 cells in a concentration-dependent manner with an EC 50 value of 0.25 (0.12–0.53) Μmol/L and 0.39 (0.27–0.57) Μmol/L at 6 h/0.59 (0.22–1.58) Μmol/L at 12 h, respectively. SKF38393 also increased the intracellular calcium level in a concentration-dependent manner with EC 50 value of 27 (8.6–70) nmol/L. Conclusion : An HEK293 cell line stably expressing human D 1 R was obtained successfuly. The study also demonstrated that the CRE-SEAP activity assay could be substituted for the cAMP accumulation assay for measuring increase in cAMP levels. Thus, both intracellular calcium measurements and the CRE-SEAP activity assay are suitable for high-throughput screening in drug research.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75187/1/j.1745-7254.2005.00017.x.pd

Dynamic distribution and tissue tropism of classical swine fever virus in experimentally infected pigs

<p>Abstract</p> <p>Background</p> <p><it>Classical swine fever </it>(CSF), caused by the <it>Classical swine fever virus </it>(CSFV), is an Office International des Epizooties (OIE) notifiable disease. However, we are far from fully understand the distribution, tissue tropism, pathogenesis, replication and excretion of CSFV in pigs. In this report, we investigated the dynamic distribution and tissue tropism of the virus in internal organs of the experimentally infected pigs using real-time RT-PCR and immunohistochemistry (IHC).</p> <p>Results</p> <p>A relative quantification real-time PCR was established and used to detect the virus load in internal organs of the experimentally infected pigs. The study revealed that the virus was detected in all 21 of the internal organs and blood collected from pigs at day 1 to day 8 post infections, and had an increasing virus load from day 1 to day 8 post infections. However, there was irregular distribution virus load in most internal organs over the first 2 days post infection. Blood, lymphoid tissue, pancreas and ileum usually contain the highest viral loads, while heart, duodenum and brain show relatively low viral loads.</p> <p>Conclusions</p> <p>All the data suggest that CSFV had an increasing virus load from day 1 to day 8 post infections in experimentally infected pigs detected by real-time RT-PCR, which was in consistent with the result of the IHC staining. The data also show that CSFV was likely to reproduce in blood, lymphoid tissue, pancreas and the ileum, while unlikely to replicate in the heart, duodenum and brain. The results provide a foundation for further clarification of the pathogenic mechanism of CSFV in internal organs, and indicate that blood, lymphoid tissue, pancreas and ileum may be preferred sites of acute infection.</p

Earthquake Forecast via Neutrino Tomography

We discuss the possibility of forecasting earthquakes by means of (anti)neutrino tomography. Antineutrinos emitted from reactors are used as a probe. As the antineutrinos traverse through a region prone to earthquakes, observable variations in the matter effect on the antineutrino oscillation would provide a tomography of the vicinity of the region. In this preliminary work, we adopt a simplified model for the geometrical profile and matter density in a fault zone. We calculate the survival probability of electron antineutrinos for cases without and with an anomalous accumulation of electrons which can be considered as a clear signal of the coming earthquake, at the geological region with a fault zone, and find that the variation may reach as much as 3% for $\bar \nu_e$ emitted from a reactor. The case for a $\nu_e$ beam from a neutrino factory is also investigated, and it is noted that, because of the typically high energy associated with such neutrinos, the oscillation length is too large and the resultant variation is not practically observable. Our conclusion is that with the present reactor facilities and detection techniques, it is still a difficult task to make an earthquake forecast using such a scheme, though it seems to be possible from a theoretical point of view while ignoring some uncertainties. However, with the development of the geology, especially the knowledge about the fault zone, and with the improvement of the detection techniques, etc., there is hope that a medium-term earthquake forecast would be feasible.Comment: 6 pages, 4 figures, 1 tabl

Real-time monitoring of airborne molecular contamination on antireflection silica coatings using surface acoustic wave technology

Real time monitoring of contamination on antireflection (AR) silica coatings in high peak power laser systems (HPLs) is critically needed in order to avoid reductions of transmission and laser damage to optical surfaces. Herein we proposed to apply a surface acoustic wave (SAW) sensor to real-time monitor trace amounts of airborne molecular contaminants (AMCs) adsorbed on the AR silica coatings. The silica coating is found to be susceptible to AMCs because of its mesoporous structure, huge surface area and polar nature. The adsorbed AMCs caused the increased mass on the silica coating of the SAW sensor, which resulted in a significant increase of its frequency shift. The fabricated sensor showed a high sensitivity of ∼-490 mm2 ng−1Hz and an excellent linearity vs. the areal density of adsorbed AMCs since the frequency shift of the sensor is linearly related to the change of mass of the silica coating

Elastic loading enhanced NH3 sensing for surface acoustic wave sensor with highly porous nitrogen doped diamond like carbon film

We proposed a surface acoustic wave (SAW) NH3 gas sensor based on nitrogen doped diamond like carbon (N-DLC) film. The N-DLC film, prepared using a microwave electron cyclotron resonance plasma chemical vapor deposition (ECR-PECVD) method, is highly porous and physically and chemically stable, and have active polar groups on its surface, which can selectively absorb polar NH3 gas molecules. These features of the film lead to the high sensitivity, low noise and excellent stability of the sensor. The sensor can achieve capabilities of in-situ monitoring NH3 in a concentration range from 100 ppb to 100 ppm with fast response (∼5 s) and recovery (∼29 s) at room temperature. The NH3 sensing mechanism is attributed to the decreased porosity of the N-DLC film caused by adsorbed NH3 molecules on its polar groups, which leads an increase of the elastic modulus of the film