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The laser transmitter and photoelectric receiver are the core modules of the detector in a laser proximity fuse, whose performance variability can affect the accuracy of target detection and identification. In particular, there is no study on the effect of detector’s component performance variability on frequency-modulated continuous-wave (FMCW) laser fuse under smoke interference. Therefore, based on the principles of particle dynamic collision, ray tracing, and laser detection, this paper builds a virtual simulation model of FMCW laser transmission with the professional particle system of Unity3D, and studies the effect of performance variability of laser fuse detector components on the target characteristics under smoke interference. Simulation results show that the difference in the performance of the fuse detector components causes the amplitude variation and peak migration of the beat signal spectrum, and the change in the visibility of the smoke can also affect the results, which indicates that the factors affecting the signal-to-noise ratio (SNR) of the echo signal are related to the smoke interference and performance variability of the detector. The proposed simulation model is supported by experimental results, which reflect the reliability of the proposed findings. Therefore, this study can be used for the optimization of the parameters in the laser fuse antismoke interference to avoid false alarms.

Surfactant polymer (SP) flooding has become an important enhanced oil recovery (EOR) technique for the high-water cut mature oilfield. Emulsification in the SP flooding process is regarded as a powerful mark for the successful application of SP flooding in the filed scale. People believe emulsification plays a positive role in EOR. This paper uses one-dimensional homogenous core flooding experiments and parallel core flooding experiments to examine the effect of emulsification on the oil recoveries in the SP flooding process. 0.3 pore volume (PV) of emulsions which are prepared using ultralow interface intension (IFT) SP solution and crude oil with stirring method was injected into core models to mimic the emulsification process in SP flooding, followed by 0.35 PV of SP flooding to flood emulsions and remaining oil. The other experiment was preformed 0.65 PV of SP flooding as a contrast. We found SP flooding can obviously enhance oil recovery factor by 25% after water flooding in both homogeneous and heterogeneous cores. Compared to SP flooding, emulsification can contribute an additional recovery factor of 3.8% in parallel core flooding experiments. But there is no difference on recoveries in homogenous core flooding experiments. It indicates that the role of emulsification during SP flooding will be more significant for oil recoveries in a heterogeneous reservoir rather than a homogeneous reservoir.

Polymer flooding is a tertiary oil recovery technology that is very suitable for the characteristics of China’s reservoirs. However, due to the fast flow rate of polymer solution in near-well zone, the shear effect of perforation blasthole and the compacted zone results in serious loss of polymer viscosity. In this paper, the polymer used in Dagang Oilfield is studied by simulation experiment through the shearing process of perforating holes, and the influence of different perforating parameters on polymer viscosity loss is analyzed, so as to provide theoretical basis for the optimization design of perforating technology in field test. The experimental results show that, the shear effect of perforation blasthole on polymer is not obvious, and the viscosity retention rate of polymer solution is greater than 96%. The size and shape of perforation blasthole have no effect on viscosity loss of polymer solution. The shear effect of compacted zone on polymer is obvious, and the viscosity retention rate of polymer solution is lower than 64% for the target block. The viscosity loss of polymer solution increases with flow rate at compacted zone, and the decrease of permeability can increase viscosity loss of polymer solution. The higher the polymer concentration is, the stronger the shear resistance is, while the higher the molecular weight is, the weaker the shear resistance is. It is suggested that perforation gun and perforation method with deep perforation depth and low compaction degree be chosen to reduce the flow rate at compacted zone and viscosity loss of polymer solution.

A novel series of Si-bearing, nearly Ni-free economical TRIP-aided duplex stainless steels (DSSs) 19Cr-5.5Mn-0.3Ni-0.2N-xSi (x = 0.5-2.5) have been prepared to investigate effect of silicon on microstructure, tensile mechanical property and high-temperature oxidation behavior. The results have shown that the designed economical alloys have a balanced ferrite/austenite dual-phase structure after suitable solution treatment. Volume of ferrite phase increases with an increase in silicon content. Ferrite deforms by dislocation slip during tensile plastic deformation process at room temperature. However, transformation-induced plasticity effect dominates deformation mode of metastable austenite phase. The designed DSS with 1.5 wt.% Si content exhibits good combination of tensile mechanical properties, exceeding 50% in elongation and 800 MPa in ultimate tensile strength, with a sequential γ → e-martensite → α′-martensite transformation in deformed austenite phase. High-temperature oxidation resistance of DSSs could be enhanced with an increase in silicon content at 1100 °C in air due to the formation of compact and continuous Cr- and Si-rich oxide film in the inner oxide film layer.

The magnetostriction of Fe-Ga alloys is significantly affected by the Ga content, which displays two positive peaks with increasing Ga concentration. This work presents a systematic structural investigation of rapidly-cooled Fe-Ga alloys by the synchrotron-based high-energy x-ray diffraction. Ga-rich clusters are evidenced for the alloys beyond the first magnetostriction peak, which is identified by the anomalous peak broadening for the (200) fundamental reflection and a shrink of lattice parameter. The generation of Ga-rich clusters in A2 matrix results in the sudden decrease of magnetostriction after the first magnetostriction peak. These findings are important for designing high-performance Fe-Ga magnetostrictive alloys by structural tuning.

In order to ensure the rationality of screed structure design and reliability of structure strength, the structural finite element model of paver screed was established with the aid of APDL language. The static and dynamic characteristics of the screed are analysed based on the finite element method. Results show that maximum principal stress of screed in the bar is 113Mpa, maximum deformation is 1.82mm, the screed meets the normal demand both in intensity and rigidity. Natural frequencies and according vibration model of screed were obtained from finite element modal analysis, the first-order natural frequency in a vibrator adjustable frequency range, so working frequency should avoid the first-order natural frequency, and the change rule of strain and stress amplitudes of screed with the vibration frequency was obtained from harmonic response analysis. The structural static and dynamic analysis of screed provides the basis for the structure design and the adjustment of construction parameters.

We investigate the soliton propagation in lead glass with rectangle boundary by the method of image beams. This method is the analogue of the electric image method which is used to solve the Poisson type equation. In the general case that the beam width is much smaller than the boundary size, the soliton formation and the soliton steering are analytically studied independently. The critical power of the soliton formation is boundary independent. The boundary effect only provides, for the soliton steering, the dynamic force which is equivalent to the force between the soliton beam and the infinite number of image beams. The closed-form steering trajectories of the solitons are in good agreement with the experimental results.

With the development of mobile computing, location-based service (LBS) is becoming an important aspect of applied research, the technology of service discovery becomes a core issue. So service matching is the most important in service discovery, also it became hot spot in researching. In the process of providing service to user, not only the quality of service should be improved, but also user’s preferences should be considered. The existing mainstream service discovery protocols in the location-oriented service were compared in this paper. Based on analysis and comparison, a new algorithm of service matching is proposed in this paper. It improves the service matching success rate through hierarchical matching and full accounting of the user’s preference, and ultimately aim to improve service quality.

Background and purpose: A prostamide analogue, bimatoprost, has been shown to be effective in reducing intraocular pressure, but its precise mechanism of action remains unclear. Hence, to elucidate the molecular mechanisms of this effect of bimatoprost, we focused on pharmacologically characterizing prostaglandin FP receptor (FP) and FP receptor variant (altFP) complexes. Experimental approach: FP receptor mRNA variants were identified by reverse transcription-polymerase chain reaction. The FP-altFP4 heterodimers were established in HEK293/EBNA cells co-expressing FP and altFP4 receptor variants. A fluorometric imaging plate reader was used to study Ca2+ mobilization. Upregulation of cysteine-rich angiogenic protein 61 (Cyr61) mRNA was measured by Northern blot analysis, and phosphorylation of myosin light chain (MLC) by western analysis. Key results: Six splicing variants of FP receptor mRNA were identified in human ocular tissues. Immunoprecipitation confirmed that the FP receptor is dimerized with altFP4 receptors in HEK293/EBNA cells co-expressing FP and altFP4 receptors. In the studies of the kinetic profile for Ca2+ mobilization, prostaglandin F2α (PGF2α) elicited a rapid increase in intracellular Ca2+ followed by a steady state phase. In contrast, bimatoprost elicited an immediate increase in intracellular Ca2+ followed by a second phase. The prostamide antagonist, AGN211335, selectively and dose-dependently inhibited the bimatoprost-initiated second phase of Ca2+ mobilization, Cyr61 mRNA upregulation and MLC phosphorylation, but did not block the action of PGF2α. Conclusion and implications: Bimatoprost lacks effects on the FP receptor but may interact with the FP-altFP receptor heterodimer to induce alterations in second messenger signalling. Hence, FP-altFP complexes may represent the underlying basis of bimatoprost pharmacology.

Background and Purpose: The prostamides (prostaglandin-ethanolamides) and prostaglandin (PG) glyceryl esters are biosynthesized by COX-2 from the respective endocannabinoids anandamide and 2-arachidonyl glycerol. Agonist studies suggest that their pharmacologies are unique and unrelated to prostanoid receptors. This concept was further investigated using antagonists. Experimental Approach: The isolated feline iris was used as a key preparation, where prostanoid FP receptors and prostamide activity co-exist. Activity at human recombinant FP and other prostanoid receptors was determined using stable transfectants. Key Results: In the feline iris, AGN 204396 produced a rightward shift of the dose-response curves for prostamide F2α and the prostamide F2α analog bimatoprost but did not block the effects of PGF2α and synthetic FP receptor agonists. Studies on human recombinant prostanoid receptors confirmed that AGN 204396 did not behave as a prostanoid FP receptor antagonist. AGN 204396 exhibited no antagonism at DP and EP1-4, but was a highly effective TP receptor antagonist. Contrary to expectation, the FP receptor antagonist AL-8810 efficaciously contracted the cat iris. AGN 204396 did not affect AL-8810 induced contractions, demonstrating that AL-8810 and AGN 204396 are pharmacologically distinct. Unlike AL-8810, the ethylamide derivate of AL-8810 was not an agonist. Al-8810 did not block prostamide F2α activity. Finally, AGN 204396 did not block PGE2-glyceryl ester activity. Conclusions and Implications: The ability of AGN 204396 to selectively block prostamide responses suggests the existence of prostamide sensitive receptors as entities distinct from receptors recognizing PGF2α and PGE2-glyceryl ester. British Journal of Pharmacology (2007) 150, 342–352. doi:10.1038/sj.bjp.0706989

The aim of glaucoma therapy is to preserve vision by reducing intraocular pressure (IOP). Following recent National Eye Institute sponsored studies, it is becoming increasingly apparent that every mmHg of extra IOP lowering counts. Bimatoprost is the newest and most effective addition to the physician's armamentarium of ocular hypotensive drugs. Direct clinical comparisons have demonstrated that it is more efficacious than the prostaglandin (PG) FP receptor agonist prodrugs, latanoprost and travoprost, as well as a β-adrenoceptor antagonist, timolol, alone or in fixed combination with the carbonic anhydrase inhibitor, dorzolamide. Moreover, patients that are refractory to latanoprost therapy may be successfully treated with bimatoprost. Such evidence provides support, at the clinical level, for the contention that bimatoprost is pharmacologically distinct from PG FP receptor agonist prodrugs. Bimatoprost is a structural analog of PGF2α-ethanolamide (prostamide F2α), which is formed from the endocannabinoid anandamide by a biosynthetic pathway involving cyclooxygenase-2 (COX-2). Their pharmacology is remarkably similar, such that bimatoprost may be regarded as a prostamide mimetic. The target receptor for bimatoprost and the prostamides appears unique and unrelated to PG- and endocannabinoid-sensitive receptors. Extensive ocular distribution/metabolism studies in non-human primates demonstrate that bimatoprost is not a prodrug, it remains essentially intact. Its profound ocular hypotensive effects may, therefore, be attributed to its prostamide-mimetic properties.

Regulator of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) for Galpha subunits and negatively regulate G protein-coupled receptor signaling. Using RGS5 gene-specific RT-PCR, we have identified a novel alternative splicing variant of RGS5 mRNA in human ocular tissues. The alternative splicing of RGS5 mRNA occurred at position +44 (GenBank NM_003617), spliced out 174 bp (+44 to +218 bp) of the coding region, and encoded an RGS5s protein with a 108 amino acid N-terminal deletion. This study is the first to document alternative splicing of an RGS5 gene. We therefore studied RGS5 and RGS5s mRNA distribution in human tissues. In the eye, RGS5s was found to be highly expressed in the ciliary body and trabecular meshwork. It was also expressed in the kidney, brain, spleen, skeletal muscle and small intestine, but was not detectable in the liver, lung, heart. RGS5s was not found in monkey and rat ocular tissues, indicating species specificity for the eye. Comparing the recombinant RGS5 and RGS5s expression in HEK293/EBNA cells, RGS5s was present almost exclusively in the cytosolic fraction, whereas RGS5 was present in both membrane and cytosolic fractions. The data suggest that the N-terminal of RGS5 may be important for protein translocation to the cell membrane. Both RGS5 and RGS5s antagonized the rapid phosphorylation of p44/42 MAP kinase induced by Galphai coupled cannibinoid receptor-1 activation. RGS5, but not RGS5s, inhibited the Ca2+ signaling initiated by activation of Galphaq coupled angiotensin II receptors (AT1) and prostaglandin FP receptors. Cotransfection of RGS5s with RGS5 resulted in the blockade of RGS5 actions with respect to inhibition of the signal transduction initiated by activation of both AT1 and FP receptor, suggesting that RGS5s may contain functional domains that compete with RGS5 in the regulation of the Galphaq coupled AT1 and FP receptors. The unique expression pattern, cellular localization and functions of RGS5s suggest that RGS5s may play a critical role in the regulation of intracellular signaling pathways.

1. Using gene chip technology, we first identified that PGF(2alpha) (FP agonist) and Butaprost (EP(2) agonist) induced about a five-fold upregulation of Nur77 mRNA expression in hFP-HEK 293/EBNA and hEP(2)-HEK293/EBNA cells. Northern Blot analysis revealed that PGF(2alpha)- and Butaprost-induced upregulation of Nur77 expression are dose- and time-dependent. 2. Both PGF(2alpha) and Butaprost upregulated Nur77 gene expression through the protein kinase C (PKC) pathway. These data are the first showing a link between EP(2) receptor stimulation and protein kinase C activation. Calcineurin was found to be involved downstream of the PKC pathway in PGF(2alpha)-induced Nur77 expression, but not in Butaprost-induced Nur77 expression. 3. We also used Nur77 as a marker gene to compare the effects of PGF(2alpha), Butaprost, and Bimatoprost (a prostamide) on Nur77 expression in human primary trabecular meshwork and ciliary smooth muscle (SM) cells, which are target cells for antiglaucoma drugs. The results showed that PGF(2alpha) and Butaprost, but not Bimatoprost, induced upregulation of Nur77 expression in human TM cells. PGF(2alpha), but not Bimatoprost, dramatically induced upregulation of Nur77 mRNA expression in human ciliary SM cells, whereas Butaprost slightly upregulated Nur77 mRNA expression in SM cells. 4. Nur77 promoter deletion analysis indicated that PGF(2alpha), but not Bimatoprost, activated Nur77 promoter-luciferase reporter in hFP-HEK 293/EBNA cells. Butaprost was less efficacious in inducing Nur77 promoter-luciferase reporter activity in hEP(2)-HEK293/EBNA cells relative to PGF(2alpha) in the comparable assay. The data for Nur77 promoter functional analysis were matched to the Northern blot analysis. 5. It appears that PGF(2alpha) and Butaprost activate Nur77 transcription mechanisms through the activation of FP and EP(2) receptor-coupled signaling pathways, whereas Bimatoprost stimulates neither FP nor EP(2) receptors.

Connective tissue growth factor (CTGF) and Cyr61 (cysteine-rich angiogenic protein 61) are members of the CCN gene family that encode multifunctional, extracellular matrix-associated signaling proteins. Because the mechanism of action of certain anti-glaucoma drugs involves extracellular matrix remodeling of ocular ciliary muscle, with a resultant increase in drainage of aqueous humor from the eye, we compared the effects of three pharmacologically distinct ocular hypotensive agents on Cyr61 and CTGF gene expression. Thus, prostaglandin F2alpha (PGF2alpha) (FP receptor agonist), Butaprost (EP2 receptor agonist), and Bimatoprost (a prostamide) were compared. Using Affymetrix gene chip technology, we first identified that PGF2alpha dramatically up-regulated Cyr61 and CTGF mRNA expression in HEK 293/EBNA cells (hFP-HEK 293/EBNA). Northern blot further confirmed the Cyr61 and CTGF up-regulation is in a dose- and time-dependent manner. PGF2alpha-induced up-regulation of Cyr61 appeared to exclusively involve the Rho pathway, and up-regulation of CTGF was via multiple intracellular pathways. Because prostamide receptors are, to date, defined only at the pharmacological level, Bimatoprost effects on Cyr61 and CTGF were studied in the isolated feline iris sphincter preparation, a tissue highly responsive to prostamides. Both PGF2alpha and Bimatoprost up-regulated Cyr61 mRNA expression in the cat iris tissue. Only PGF2alpha up-regulated CTGF mRNA expression in the cat iris. Therefore, PGF2alpha and Bimatoprost appear to interact with different receptors populations in the cat iris, according to their markedly different effects on CTGF. Activation of prostaglandin EP2 receptors (Gs-coupled) also up-regulated Cyr61 but not CTGF mRNA expression in the isolated cat iris. Similar data were observed in human primary ciliary smooth muscle cells. Thus, despite quite different signal transduction pathways, FP receptor stimulation up-regulates CTGF and Cyr61. The prostamide analog Bimatoprost and an EP2-selective agonist affects only Cyr61.

Apolipoprotein E (apoE) and apoJ are lipid carriers produced in the brain primarily by glial cells. A variety of glial-activating stimuli induce a parallel upregulation of both apolipoproteins expression in vivo and in vitro. To further characterize the cell type and mechanisms by which apoE and apoJ expression are upregulated in activated glia, mixed glial cultures from neonatal rat cortex were treated with the endotoxin lipopolysaccharide (LPS). LPS induced dose-dependent increases in apoJ and decreases in apoE expression and secretion with maximum effects at 1-10 ng/mL and 0.1-1 microg/mL, respectively. Experiments with enriched astroglial and microglial cultures demonstrated that apoE and apoJ expression are predominantly microglial and astroglial, respectively. Given the pivotal role that nuclear factor-kappa B (NF-kappa B) plays in glial activation, we assessed its possible role in mediating apoE and apoJ expression by activated glia. LPS robustly increased NF-kappa B activation in mixed glial cultures. Two NF-kappa B inhibitors, aspirin (10 mM) and MG-132 (0.1 microM), blocked basal apoE and apoJ secretion as well as LPS-induced apoJ secretion. These data demonstrate that glial apoE and apoJ expression are independently regulated by LPS in microglia and astroglia, respectively, and that activated microglia are the predominant source of apoE in mixed glial cultures. The transcription factor NF-kappa B appears to be a critical mediator of LPS-stimulated apoJ expression from astroglia.

Replacement of the carboxylic acid group of prostaglandin (PG) F(2alpha) with a nonacidic moiety, such as hydroxyl, methoxy, or amido, results in compounds with unique pharmacology. Bimatoprost (AGN 192024) is also a pharmacologically novel PGF(2alpha) analog, where the carboxylic acid is replaced by a neutral ethylamide substituent. Bimatoprost potently contracted the feline lung parenchymal preparation (EC(50) value of 35-55 nM) but exhibited no meaningful activity in a variety of PG-sensitive tissue and cell preparations. Its activity seemed unrelated to FP receptor stimulation according to the following evidence. 1) Bimatoprost exhibited no meaningful activity in tissues and cells containing functional FP receptors. 2) Bimatoprost activity in the cat lung parenchyma is not species-specific because its potent activity in this preparation could not be reproduced in cells stably expressing the feline FP receptor. 3) Radioligand binding studies using feline and human recombinant FP receptors exhibited minimal competition versus [(3)H]17-phenyl PGF(2a) for Bimatoprost. 4) Bimatoprost pretreatment did not attenuate PGF(2alpha)-induced Ca(2+) signals in Swiss 3T3 cells. 5) Regional differences were apparent for Bimatoprost but not FP agonist effects in the cat lung. Bimatoprost reduced intraocular pressure in ocular normotensive and hypertensive monkeys over a 0.001 to 0.1% dose range. A single-dose and multiple-dose ocular distribution/metabolism studies using [(3)H]Bimatoprost (0.1%) were performed. Within the globe, bimatoprost concentrations were 10- to 100-fold higher in anterior segment tissues compared with the aqueous humor. Bimatoprost was overwhelmingly the predominant molecular species identified at all time points in ocular tissues, indicating that the intact molecule reduces intraocular pressure.

Opioid compounds have potent analgesic and euphoric properties. They act with specific cell-membrane receptors which have been pharmacologically defined into three major classes, mu, kappa and delta. These receptors are highly regulated with respect to their gene expression, resulting in a temporally and spatially specific pattern of distribution for each receptor. To characterize the promoter sequence of the mu opioid receptor (MOR) gene, a mouse genomic DNA library was screened under high stringency with a rat MOR (MOR-1) cDNA probe and genomic sequences for the mouse MOR gene were isolated. From one genomic clone, a 2.3-kb EcoRI fragment, which hybridized to the 5'-end of the rat MOR-1 cDNA probe, was subcloned and sequenced. This fragment contains 1.3 kb of sequence upstream of the initiation codon, extends downstream through exon 1 and includes a portion of intron 1. Primer extension analysis using mouse brain poly (A)+ RNA identified a transcription initiation site 793 bp upstream from the translation start site. Chimeric constructs of mouse MOR deletion fragments fused to a luciferase reporter gene were transfected into a human neuroblastoma cell line, SK-N-SH, which constitutively expresses endogenous MOR. These transient expression studies indicated that the 0.2-kb region upstream from the transcription initiation site possesses a functional promoter, which directs the expression of the reporter gene in vitro and may possess promoter activity for the mouse MOR gene in vivo.

We investigate the propagation of the spatial soliton in cylindrical strongly nonlocal media by a novel method of image beam of light. The effect of the boundary on the soliton acting as the dynamic force for the soliton steering is equivalent to the force between the soliton beam and the image beam. The trajectory of the soliton is analytically studied which is in good agreement with the experimental results., Comment: 3 Pages, 3 figures

The discovery of anandamide and 2-arachidonyl glycerol (2-AG) as naturally occurring mammalian endocannabinoids has had important and wide-reaching therapeutic implications. This, to a large extent, ensues from the complexity of endocannabinoid biology. One facet of endocannabinoid biology now receiving increased attention is the cyclo-oxygenase-2 (COX-2) derived oxidation products. Anandamide and 2-AG are oxidized to a range of PG-ethanolamides and PG-glyceryl esters that closely approaches that of the prostaglandins (PGs) formed from arachidonic acid. The pharmacology of these electrochemically neutral PG-ethanolamides (prostamides) and PG-glyceryl esters appears to be unique. No meaningful interaction with natural or recombinant prostanoid receptors is apparent. Nevertheless, in certain cells and tissues, prostamides and PG-glyceryl esters exert potent effects. The recent discovery of selective antagonists for the putative prostamide receptor has been a major advance in further establishing prostamide pharmacology as an entity distinct from prostanoid receptors. Since discovery of the prototype prostamide antagonist (AGN 204396), rapid progress has been made. The latest prostamide antagonists (AGN 211334-6) are 100 times more potent than the prototype and are, therefore, sufficiently active to be used in living animal studies. These compounds will allow a full evaluation of the role of prostamides in health and disease. To date, the only therapeutic application for prostamides is in glaucoma. The prostamide analog, bimatoprost, being the most effective ocular hypotensive drug currently available. Interestingly, PGE(2)-glyceryl ester and its chemically stable analog PGE(2)-serinolamide also lower intraocular pressure in dogs. Nevertheless, the therapeutic future of PGE(2)-glyceryl ester is more likely to reside in inflammation.

Regulator of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) for Galpha subunits and negatively regulate G protein-coupled receptor signaling. Using RGS5 gene-specific RT-PCR, we have identified a novel alternative splicing variant of RGS5 mRNA in human ocular tissues. The alternative splicing of RGS5 mRNA occurred at position +44 (GenBank NM_003617), spliced out 174 bp (+44 to +218 bp) of the coding region, and encoded an RGS5s protein with a 108 amino acid N-terminal deletion. This study is the first to document alternative splicing of an RGS5 gene. We therefore studied RGS5 and RGS5s mRNA distribution in human tissues. In the eye, RGS5s was found to be highly expressed in the ciliary body and trabecular meshwork. It was also expressed in the kidney, brain, spleen, skeletal muscle and small intestine, but was not detectable in the liver, lung, heart. RGS5s was not found in monkey and rat ocular tissues, indicating species specificity for the eye. Comparing the recombinant RGS5 and RGS5s expression in HEK293/EBNA cells, RGS5s was present almost exclusively in the cytosolic fraction, whereas RGS5 was present in both membrane and cytosolic fractions. The data suggest that the N-terminal of RGS5 may be important for protein translocation to the cell membrane. Both RGS5 and RGS5s antagonized the rapid phosphorylation of p44/42 MAP kinase induced by Galphai coupled cannibinoid receptor-1 activation. RGS5, but not RGS5s, inhibited the Ca2+ signaling initiated by activation of Galphaq coupled angiotensin II receptors (AT1) and prostaglandin FP receptors. Cotransfection of RGS5s with RGS5 resulted in the blockade of RGS5 actions with respect to inhibition of the signal transduction initiated by activation of both AT1 and FP receptor, suggesting that RGS5s may contain functional domains that compete with RGS5 in the regulation of the Galphaq coupled AT1 and FP receptors. The unique expression pattern, cellular localization and functions of RGS5s suggest that RGS5s may play a critical role in the regulation of intracellular signaling pathways.

Given the relatively recent introduction, established effectiveness, and widespread clinical use of selective serotonin reuptake inhibitors (SSRIs) (1), one might legitimately ask, “Why do we need new antidepressant agents?” Indeed, the SSRIs and their less-selective predecessors, the tricyclic antidepressants and monoamine oxidase inhibitors, are effective in approx 65–70% of patients when systematically investigated in “double-blind” placebo-controlled trials (2,3). Moreover, patients who fail to respond to one antidepressant often respond to another compound, either from the same or different “class” (3). It has also been estimated that at least 80% of depressed patients will eventually respond to one or more antidepressants, and this may be greater than 90% for patients treated with electroconvulsive therapy (ECT) (4).

Apolipoprotein E (apoE) and apoJ are lipid carriers produced in the brain primarily by glial cells. A variety of glial-activating stimuli induce a parallel upregulation of both apolipoproteins expression in vivo and in vitro. To further characterize the cell type and mechanisms by which apoE and apoJ expression are upregulated in activated glia, mixed glial cultures from neonatal rat cortex were treated with the endotoxin lipopolysaccharide (LPS). LPS induced dose-dependent increases in apoJ and decreases in apoE expression and secretion with maximum effects at 1-10 ng/mL and 0.1-1 microg/mL, respectively. Experiments with enriched astroglial and microglial cultures demonstrated that apoE and apoJ expression are predominantly microglial and astroglial, respectively. Given the pivotal role that nuclear factor-kappa B (NF-kappa B) plays in glial activation, we assessed its possible role in mediating apoE and apoJ expression by activated glia. LPS robustly increased NF-kappa B activation in mixed glial cultures. Two NF-kappa B inhibitors, aspirin (10 mM) and MG-132 (0.1 microM), blocked basal apoE and apoJ secretion as well as LPS-induced apoJ secretion. These data demonstrate that glial apoE and apoJ expression are independently regulated by LPS in microglia and astroglia, respectively, and that activated microglia are the predominant source of apoE in mixed glial cultures. The transcription factor NF-kappa B appears to be a critical mediator of LPS-stimulated apoJ expression from astroglia.

Two transcription initiation sites have been identified in the mouse μ-opioid receptor (MOR) gene at approximately −793 and −268 upstream of the translation start site. To test if the MOR gene contains two functional promoters, chimeric constructs of mouse MOR deletion fragments, fused to a luciferase reporter gene, were transiently transfected into the neuroblastoma cell line SK-N-SH, a MOR-expressing cell line, and two MOR-non-expressing cell lines. Results from transient transfection assays confirmed the existence of two functional independent promoters in the mouse MOR gene, and also revealed that the region from −1337 to −93 does not contain all the elements necessary to confer tissue-specific expression of the MOR gene.

In a previous study we showed that a region from -182 to +10 bp in the mouse mu-opioid receptor (MOR) promoter exhibited strong promoter activity. To identify protein-DNA interactions in this fragment, gel shift and DNase I footprint analyses were performed using nuclear extracts from mouse brain and the human neuroblastoma cell line, SK-N-SH. Two regions, nucleotide (nt) -121 to -100 and nt -42 to -22, were identified as being specific protein binding sites. The protein-DNA interaction in the nt -42 to -22 region was characterized in detail in this study. Methylation interference analysis of this region showed that nuclear protein from SK-N-SH cells contracted nucleotides within the sequence ATG-CAAAT, which is a binding motif for octamer trans-acting factors. An octamer-1 (Oct-1)-specific antibody super-shifted the protein-DNA complex in a gel shift assay. A UV cross-linking experiment showed that a nuclear protein, whose molecular weight is similar to that of the Oct-1 factor, bound to the octamer element in the nt -42 to -22 region. Mutagenesis of four base pairs within the octamer cis-acting element eliminated the specific protein binding in vitro. When the MOR-luciferase reporter construct (-182 to +10 bp) with the same four base pairs mutated was transiently transfected into SK-N-SH cells, a 200% increase in transcriptional activity was observed. Collectively, these data suggest that Oct-1 is binding to the octamer motif in the MOR gene and negatively modulating MOR gene expression.

Because alcoholic hepatitis and cirrhosis, well-known complications of alcohol abuse, do not occur in all alcoholics, genetic factors such as differences in alcohol-metabolizing enzymes may play a role in the development of alcoholic liver disease. Cytochrome P450IIE1 catalyzes the oxidation of ethanol, producing acetaldehyde and free radicals capable of reacting with and peroxidizing cell membranes. Polymorphisms have been identified in the 5'-flanking region of the P450IIE1 gene that may alter the transcriptional activity of the gene. In this study, we analyzed the P450IIE1 genotypes at the polymorphic PstI and RsaI restriction enzyme sites in 53 Caucasians with severe alcoholic liver disease to determine if there is an association between these polymorphisms and alcoholic liver disease. Subjects that tested positive for the hepatitis C virus were eliminated from the study. To identify the type A (homozygous for the c1 gene), type B (heterozygous for the c1 and c2 genes), and type C (homozygous for the c2 gene) genotypes at the P450IIE1 locus, DNA encompassing the polymorphisms was amplified by polymerase chain reaction, slot-blotted, and probed with allele-specific oligonucleotides. Allele frequencies for the c1 allele were 0.95 for alcoholics with severe liver disease, 0.95 for alcoholics without liver disease, and 0.98 for the general population. No differences in allele frequencies between alcoholic patients with severe liver disease and alcoholics without liver disease were observed.