Cationic oligonucleotides can mediate specific inhibition of gene expression in Xenopus oocytes.

Base-specific hydrogen bonding between an oligonucleotide and the purines in the major groove of a DNA duplex provide an approach to selective inhibition of gene expression. Oligonucleotide-mediated triplex formation in vivo may be enhanced by a number of different chemical modifications. We have previously described an in vitro analysis of triplex formation using oligonucleotides containing internucleoside phosphate linkages modified with the cation N , N -diethyl-ethylenediamine (DEED). When compared with unmodified oligonucleotides of identical base composition, DEED-modified oligonucleotides were better able to form DNA triplexes under conditions that approximate the pH, magnesium and potassium levels found in vivo . Here we report the ability of DEED-modified oligonucleotides to inhibit the expression of plasmid DNA injected into Xenopus oocytes. Inhibition is specific to plasmids containing a triplex formation target and sensitive to sequence alteration in the triplex forming target site. Inhibition of gene expression was nearly complete when oligonucleotide and plasmid were mixed together prior to injection. Inhibition was partial when oligonucleotide was injected first and not evident when plasmid was injected and allowed to form chromatin prior to oligonucleotide injection. Thus, access to DNA is a determining factor in effective triplex inhibition of gene expression.     

Adenosine A(1) receptors mediate plasma exudation from the oral mucosa.

The purpose of this study was to pharmacologically characterize the adenosine receptor subtype(s) that mediates adenosine-induced increases in macromolecular efflux from the intact hamster cheek pouch. Using intravital microscopy, we found that 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX), a selective adenosine receptor-1 antagonist, but not 3,7-dimethyl-1-propargylxanthine (DMPX), a selective adenosine receptor-2 antagonist, significantly attenuated adenosine-induced leaky site formation and increased clearance of fluorescein isothiocyanate-labeled dextran (molecular mass, 70 kDa) from the intact hamster cheek pouch (P < 0.05). Both compounds had no significant effects on bradykinin-induced responses. Nanomolar concentrations of R(-)-N(6)-(2-phenylisopropyl)-adenosine [R(-)-PIA], a selective adenosine A(1) agonist, evoked significant, concentration-dependent increases in macromolecular efflux. This response was significantly attenuated by PACPX but not by DMPX. In contrast, CGS-21680, a selective adenosine A(2) agonist, increased macromolecular efflux but only at micromolar concentrations. This response was significantly attenuated by DMPX but not by PACPX. Suffusion of nitroglycerin had no significant effects on R(-)-PIA- and CGS-21680-induced responses. In addition, suffusion of N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, had no significant effects on adenosine-induced responses. Indomethacin had no significant effects on adenosine-, R(-)-PIA-, and CGS-21680-induced increases in macromolecular efflux. Collectively, these data indicate that adenosine increases macromolecular efflux from the intact hamster cheek pouch by stimulating high-affinity adenosine A(1) receptors in a specific, nitric oxide- and prostaglandin-independent fashion.     

Amylin receptors mediate the anorectic action of salmon calcitonin (sCT)

The teleost salmon calcitonin (sCT), but not mammalian CT, shows similar biologic actions in the skeletal muscle as amylin and calcitonin gene-related peptide (CGRP). The peptides have also been shown to reduce food intake in rams. Because sCT, but not amylin, binds irreversibly to amylin binding sites, the aim of the present study was to compare the anorectic potency of both peptides. To determine whether sCT reduces food intake through interaction with amylin binding sites, we also tested whether appropriate antagonists (CORP 8-37, AC 187) attenuate the anorectic effect of sCT. Finally, we wanted to know whether rat calcitonin (rCT) and sCT reduce food intake to the same extent. Peptides were injected intraperitoneally at dark onset in 24 h food-deprived rats. At doses of 5 or 0.5 microg/kg, the anorectic effect of sCT was more potent and lasted much longer (e.g. 5 microg/kg: sCT > 10 h; amylin approx. 2 h) than that of amylin. Both CORP 8-37 and AC 187 (10 microg/kg) markedly reduced the anorectic action of sCT (0.5 microg/kg). In contrast to sCT, rCT (0.5 microg/kg) had no effect on food intake. It is concluded that sCT s anorectic effect is partly mediated by amylin receptors. Irreversible binding of sCT to amylin receptors may lead to a stronger and prolonged effect in comparison to amylin due to a sustained activation of the binding sites. Similar to other actions of CTs, the anorectic potency of sCT in rats was higher than that of mammalian (rat) CT. This agrees with binding profiles of amylin, sCT, and rCT at amylin binding sites as observed in in vitro studies.     

Collagen receptors mediate early events in the attachment of epithelial (MDCK) cells

Summary Madin-Darby canine kidney (MDCK) cells kept in suspension culture for 12–15 hr displayed high-affinity binding sites for¹²⁵I-lathyritic (soluble) collagen (120,000/cell,K _D =30nm) and preferred collagens types I and IV over laminin or fibronectin as substrates during the first hour of attachment. On the other hand, after 4 hr, attachment to all four substrates was equally efficient. Upon challenge with a collagen substrate, the high-affinity sites were rapidly recruited on it (T1/2=6 min). Their occupancy by soluble collagen triggered the exocytosis of a second large population of low-affinity collagen binding sites that included laminin and seems to be involved in a second cell-attachment mechanism. These results are compatible with a twostep model of MDCK cell attachment to the substrate: first, via high-affinity collagen binding sites, and second, via laminin of cellular origin.     

5-HT(3) receptors mediate inflammation-induced unmasking of functional tachykinin responses in vitro.

Exogenously applied tachykinins produce no measurable electrophysiological responses in the somata of vagal afferent neurons [nodose ganglion neurons (NGNs)] isolated from naive guinea pigs. By contrast, after in vitro antigen challenge of nodose ganglia from guinea pigs immunized with chick ovalbumin, approximately 60% (53 of 89) of NGNs were depolarized an average of 13 +/- 1.2 mV by substance P (SP; 100 nM; n = 53). Receptor antagonists and enzyme inhibitors were utilized to screen a number of mast cell-derived mediators for their role in the uncovering or "unmasking" of functional tachykinin receptors after antigen challenge. Two chemically distinct 5-hydroxytryptamine-3-receptor antagonists significantly reduced the percentage of NGNs displaying depolarizing SP responses. Treatment with Y-25130 (1 or 10 microM) or tropisetron (1 microM) 15 min before and during antigen challenge reduced the percentage of SP-responsive neurons to approximately 20 and approximately 15% respectively. These results suggest that activation of 5-hydroxytryptamine-3 receptors plays an integral role in the unmasking of functional tachykinin receptors after specific antigen challenge of nodose ganglia. The mediator(s) underlying tachykinin-receptor unmasking in the remainder of the NGNs has yet to be characterized. However, it does not appear to be histamine, prostanoids, or peptidoleukotrienes.     

Dopamine D(2) receptors mediate amylin's acute satiety effect

The anorectic effect of the pancreatic peptide amylin has been established in numerous studies. Here, we investigated the influence of a pretreatment with dopamine (DA) D(1)- and D(2)-receptor antagonists on the anorectic effect of intraperitoneally injected amylin in rats fed a medium-fat (18% fat) diet. In 24-h food-deprived rats, pretreatment with the DA D(2)-receptor antagonist raclopride [100 microg/kg (0.2 micromol/kg) ip] significantly attenuated amylin's (5 microg/kg ip) anorectic effect, whereas raclopride alone had no effect on food intake [i.e., food intakes 1 h after injection were (n = 12): NaCl/NaCl 7.3 +/- 0.5 g; NaCl/amylin 3.9 +/- 0.6; raclopride/NaCl 7.7 +/- 0.7; raclopride/amylin 5.6 +/- 0.7]. Pretreatment with another DA D(2) receptor antagonist, sulpiride [50 mg/kg (154 micromol/kg) ip], similarly reduced amylin's satiety effect, whereas pretreatment with the DA D(1)-receptor antagonist SCH-23390 [10 microg/kg (0.03 micromol/kg) ip] did not influence amylin's effect. SCH-23390, however, completely blocked the anorexia induced by D-amphetamine (0.3 mg/kg ip). These results suggest that, under the present feeding conditions, the dopaminergic system mediates part of amylin's inhibitory effect on feeding in rats when administered intraperitoneally. This seems to involve DA D(2) receptors but not D(1) receptors.     

Two distinct sequence elements mediate retroviral gene expression in embryonal carcinoma cells.

Moloney murine leukemia virus (M-MuLV) and M-MuLV-derived retroviral vectors are not expressed in early mouse embryos or in embryonal carcinoma cells. M-MuLV-derived mutants or M-MuLV-related variants which transduce the neomycin phosphotransferase gene can, however, induce drug resistance in embryonal carcinoma cells with high efficiency. In this study we investigated the sequences critical for retroviral gene expression in two different embryonal carcinoma cell lines, F9 and PCC4. We show that two synergistically acting sequence elements mediate expression in embryonal carcinoma cells. One of these is located within the U3 region of the viral long terminal repeat, and the second one is in the 5' untranslated region of the retrovirus. The latter element, characterized by a single point mutation, affects the level of stable RNA in infected cells, suggesting a regulatory mechanism similar to that of human immunodeficiency virus in human T cells.     

Endogenously expressed estrogen receptors mediate neuroprotection in hippocampal cells (HT22)

Discovery of estrogen receptors (ER) in the central nervous system and the ability of estrogens to modulate neural circuitry and act as neurotrophic factors, suggest a therapeutic role of this steroid. To gain better understanding of the specificity and cellular mechanisms involved in estrogen-mediated neuroprotection, a mouse hippocampal neuronal cell line (HT22) was evaluated. Earlier reports indicated this cell line was devoid of ERs. Contrary to these findings, characterization of HT22 cells using RT-PCR, immunoblot, immunocytochemical, and radioligand binding techniques revealed endogenous expression of ER. The predominant subtype appeared to be ERalpha with functional activity confirmed using an ERE-tk-luciferase assay. The ability of an ER antagonist, ICI-182780, to block the neuroprotective effects of estrogens confirmed ER was involved mechanistically in neuroprotection. In conclusion, HT22 cells express functional ERalpha or a closely related ER enabling this cell line to be used to profile estrogens for neuroprotective properties acting via an ER-dependent mechanism.     

Serotonin(2) receptors mediate respiratory recovery after cervical spinal cord hemisection in adult rats.

The aim of the present study was to specifically investigate the involvement of serotonin [5-hydroxytryptamine (5-HT(2))] receptors in 5-HT-mediated respiratory recovery after cervical hemisection. Experiments were conducted on C(2) spinal cord-hemisected, anesthetized (chloral hydrate, 400 mg/kg ip), vagotomized, pancuronium- paralyzed, and artificially ventilated female Sprague-Dawley rats in which CO(2) levels were monitored and maintained. Twenty-four hours after spinal hemisection, the ipsilateral phrenic nerve displayed no respiratory-related activity indicative of a functionally complete hemisection. Intravenous administration of the 5-HT(2A/2C)-receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) induced respiratory-related activity in the phrenic nerve ipsilateral to hemisection under conditions in which CO(2) was maintained at constant levels and augmented the activity induced under conditions of hypercapnia. The effects of DOI were found to be dose dependent, and the recovery of activity could be maintained for up to 2 h after a single injection. DOI-induced recovery was attenuated by the 5-HT(2)-receptor antagonist ketanserin but not with the 5-HT(2C)-receptor antagonist RS-102221, suggesting that 5-HT(2A) and not necessarily 5-HT(2C) receptors may be involved in the induction of respiratory recovery after cervical spinal cord injury.     

Spinal GABA(A) receptors do not mediate the sympathetic baroreceptor reflex in the rat

Activation of baroreceptors causes efferent sympathetic nerve activity (SNA) to fall. Two mechanisms could account for this sympathoinhibition: disfacilitation of sympathetic preganglionic neurons (SPN) and/or direct inhibition of SPN. The roles that spinal GABA and glycine receptors play in the baroreceptor reflex were examined in anesthetized, paralyzed, and artificially ventilated rats. Spinal GABA(A) receptors were blocked by an intrathecal injection of bicuculline methiodide, whereas glycine receptors were blocked with strychnine. Baroreceptors were activated by stimulation of the aortic depressor nerve (ADN), and a somatosympathetic reflex was used as control. After an intrathecal injection of vehicle, there was no effect on any measured variable or evoked reflex. In contrast, bicuculline caused a dose-dependent increase in arterial pressure, SNA, phrenic nerve discharge, and it significantly facilitated the somatosympathetic reflex. However, bicuculline did not attenuate either the depressor response or sympathoinhibition evoked after ADN stimulation. Similarly, strychnine did not affect the baroreceptor-induced depressor response. Thus GABA(A) and glycine receptors in the spinal cord have no significant role in baroreceptor-mediated sympathoinhibition.     

Serotonin 5-HT(7) receptors mediate relaxation of porcine pial veins

Isolated porcine pial veins in the presence of active muscle tone have been shown to exhibit rhythmic contractions (RC) that are inhibited by serotonin (5-HT) in a concentration-dependent manner. The 5-HT inhibition of RC is mediated by an as yet unidentified 5-HT receptor subtype located on the vascular smooth muscle. 5-carboxamidotryptamine, which is a potent but nonselective agonist at 5-HT(7) receptors, has been shown to be the most potent inhibitor of RC in porcine pial veins. Therefore, the present study was designed to determine if the 5-HT-mediated inhibition of RC in pial veins is mediated by 5-HT(7) receptors and if 5-HT(7) receptor mRNA is expressed in endothelium-denuded pial veins; the study was done with the use of an in vitro tissue bath and RT-PCR techniques. Our findings indicated that 5-HT inhibition of RC in porcine pial veins was prevented by 5-HT(7)-receptor antagonists (clozapine, pimozide, and LY-215840) in a concentration-dependent manner. Furthermore, a strong PCR signal for the 5-HT(7) receptor was consistently detected in endothelium-denuded pial veins. Sequence analysis of the amplified products confirmed their high degree of homology with the porcine and/or human 5-HT(7)-receptor gene. Taken together, these data suggest that the 5-HT-induced inhibition of RC in porcine pial veins is at least in part mediated by 5-HT(7) receptors located on the venous smooth muscle.     

大鼠中央杏仁核5-HT3受体参与胸腺功能调制

本研究旨在探讨大鼠中央杏仁核(central amygdala,CeA)内5-HT3受体激动之后,对丝裂原刀豆球蛋白A(concanavalin A,ConA)刺激的胸腺细胞增殖反应的影响,及其潜在的神经内分泌调节环路.分别经大鼠腹腔(intraperitoneal,i.p.)、双侧侧脑室(intracerebroventricle,i.c.v.)和双侧CeA(intracentral amygdala,i.c.a.)注射选择性5-HT3受体激动剂1-phenylbiguanide(PBG),同时制备正常大鼠胸腺细胞悬液与不同浓度PBG(1×10-8～1×10-5 mol/L)体外共同孵育.经MTT法测定显示,无论有无ConA刺激,正常大鼠离体胸腺细胞在与PBG(1×10-8～1×10-5 mol/L)体外共同孵育时其增殖反应均不受后者影响;PBG i.p.(每天0.5 mg/kg,连续5 d)对ConA刺激的胸腺细胞的增殖反应亦无影响,而PBG i.c.v.(每天10 μg/侧,连续5 d)则显著增强之;当PBG i.c.a.(每天1.0 μg/侧,1 d或连续3、5、7 d)时,ConA刺激的胸腺细胞的增殖反应于给药后第1天即开始增强且日益显著,第5天达到高峰,第7天则趋于减弱.在给予PBG 5 min前相同给药部位先给予5-HT3受体拮抗剂tropisetvon(TRP)预处理可逆转PBG的促胸腺细胞增殖效应.免疫组织化学SABC法检测显示,PBG(1.0 μg/侧,i.c.a.)单次给药后各脑区可相继出现大量c-Fos阳性细胞(CeA1 h;海马及皮层1～2 h;下丘脑4 h;中脑导水管周围灰质8 h),并迅速达到各自高峰(CeA1 h;海马及皮层2 h;下丘脑4 h),与相应的生理盐水对照组及TRP预处理组相比均有显著性差异.随后,这一表达在各脑区中逐步减弱并消失(CeA4 h;海马、皮层及下丘脑8 h).由此推论,大鼠CeA内5-HT3受体至少可部分通过边缘系统-皮层-下丘脑-中脑导水管周围灰质这一神经内分泌环路调制胸腺细胞功能.     

Glutamate AMPA/kainate receptors, not GABA(A) receptors, mediate estradiol-induced sex differences in the hypothalamus

Sex differences in brain morphology underlie physiological and behavioral differences between males and females. During the critical perinatal period for sexual differentiation in the rat, gonadal steroids act in a regionally specific manner to alter neuronal morphology. Using Golgi-Cox impregnation, we examined several parameters of neuronal morphology in postnatal day 2 (PN2) rats. We found that in the ventromedial nucleus of the hypothalamus (VMN) and in areas just dorsal and just lateral to the VMN that there was a sex difference in total dendritic spine number (males greater) that was abolished by treating female neonates with exogenous testosterone. Dendritic branching was similarly sexually differentiated and hormonally modulated in the VMN and dorsal to the VMN. We then used spinophilin, a protein that positively correlates with the amount of dendritic spines, to investigate the mechanisms underlying these sex differences. Estradiol, which mediates most aspects of masculinization and is the aromatized product of testosterone, increased spinophilin levels in female PN2 rats to that of males. Muscimol, an agonist at GABA(A) receptors, did not affect spinophilin protein levels in either male or female neonates. Kainic acid, an agonist at glutamatergic AMPA/kainate receptors, mimicked the effect of estradiol in females. Antagonizing AMPA/kainate receptors with NBQX prevented the estradiol-induced increase in spinophilin in females but did not affect spinophilin level in males.     

Proteinase-activated receptors (PARs) in infection and inflammation in the gut

Proteinases have been shown to act as signaling molecules that are able to send specific signals to cells through the activation of proteinase-activated receptors (PARs). Those receptors which are expressed in a wide variety of cells in the gastrointestinal tract are considered as "emergency" mechanisms, particularly involved in inflammatory responses of the gut. Depending on the cell types of the gut in which PARs are activated, their activation interacts with all aspects of the gut physiology: motility, barrier function, transports, innate immune response, sensory functions, and even proliferation. A growing body of evidences discussed here suggests that these receptors, and the proteinases that activate them, are important mediators of the innate immune response of the gut and could play a major role in chronic inflammatory states of the gut (inflammatory bowel diseases), or infectious diseases.     

Siglec-5 (CD170) can mediate inhibitory signaling in the absence of immunoreceptor tyrosine-based inhibitory motif phosphorylation

Siglec-5 (CD170) is a member of the recently described human CD33-related siglec subgroup of sialic acid binding Ig-like lectins and is expressed on myeloid cells of the hemopoietic system. Similar to other CD33-related siglecs, Siglec-5 contains two tyrosine-based motifs in its cytoplasmic tail implicated in signaling functions. To investigate the role of these motifs in Siglec-5-dependent signaling, we used transfected rat basophil leukemia cells as a model system. Tyrosine phosphorylation of Siglec-5 led to recruitment of the tyrosine phosphatases SHP-1 and SHP-2, as seen in both pull-down assays and microscopy. Siglec-5 could efficiently inhibit FcepsilonRI-mediated calcium fluxing and serotonin release after co-cross-linking. Surprisingly, a double tyrosine to alanine mutant of Siglec-5 could still mediate strong inhibition of serotonin release in the absence of detectable tyrosine phosphorylation, whereas a double tyrosine to phenylalanine mutant lost all inhibitory activity. In comparison, suppression of Siglec-5-dependent adhesion to red blood cells was reversed by either tyrosine to alanine or tyrosine to phenylalanine mutations of the membrane proximal tyrosine-based motif. Using an in vitro phosphatase assay with synthetic and recombinant forms of the cytoplasmic tail, it was shown that a double alanine mutant of Siglec-5 had weak, but significant SHP-1 activating properties similar to those of wild type, non-phosphorylated cytoplasmic tail, whereas a double phenylalanine mutant was inactive. These findings establish that Siglec-5 can be classified as an inhibitory receptor with the potential to mediate SHP-1 and/or SHP-2-dependent signaling in the absence of tyrosine phosphorylation.     

Aggregation of hapten-bearing liposomes mediated by specific antibodies.

We studied specific membrane-membrane interactions mediated by ligand-receptor binding in a model system, which consisted of (a) FG3P, the fluorescein hapten attached to a phospholipid by a peptidyl spacer as described previously (Petrossian, A., A.B. Kantor, and J.C. Owicki. 1985. J. Lipid Res. 26:767-773), (b) antifluorescein monoclonal antibodies (MAbs), and (c) phospholipid vesicles (liposomes) into which the FG3P was incorporated. The aggregation of the hapten-bearing liposomes by four MAbs was studied by differential centrifugation. The ability of the MAbs to induce vesicle aggregation varied considerably and correlated inversely with affinity. Aggregation by one of the MAbs was studied in more detail by turbidimetry and freeze-fracture electron microscopy of samples frozen throughout the course of the aggregation. Rapid freezing was achieved with a double propane-jet apparatus. The aggregate morphologies and the time evolution of the aggregate size distribution were obtained from the two-dimensional fracture views with a stereological correction. The aggregation kinetics were simulated by considering dynamical aggregation according to a mass-action model with two parameters, the rate constants for antibody-mediated vesicle aggregation and disaggregation. Both rate constants were orders of magnitude lower than the rate constants for the corresponding interactions of antibodies with haptens either in solution or on vesicles under nonaggregating conditions.     

Increase of retroviral infection in vitro by the binding of antiretroviral antibodies.

Monoclonal antiretrovirus antibodies were assayed for their ability to influence retrovirus infection in vitro. Some antibodies increased murine cell infection by an ecotropic virus and both murine and human cell infection by an amphotropic virus. The stability of these viruses was not modified, suggesting that antibody-virus complexes may be more infectious than free virus particles.