Parasite excretory-secretory proteins elicit TRIF dependent CXCL1 and IL-6 mediated allergic inflammation.

Currently, little information is available regarding innate immunity to helminthic parasite infection. In this study, we isolated the excretory-secretory (ES) proteins from Anisakis simplex (sea mammal intestinal parasite) third stage larva. We determined that the levels of IL-17 in the lung and lung draining lymph node of mice were increased sixfold as a result of intranasal treatment with ES proteins. The ES protein treatment elicited pro-inflammatory cytokine and chemokine secretion (especially IL-6 and CXCL1) from mouse lung epithelial cell line and primary lung epithelial cells. In addition, the expression of IL-6 and CXCL1 in mouse embryonic fibroblast (MEF) cells was significantly increased by the ES protein treatment, but we did not detect these effects in the TRIF(-/-) MEF cells. These elevations of IL-6 and CXCL1 expression were also not diminished by RNase treatment. In conclusion, the ES proteins of helminthic parasite larva may elicit TRIF dependent pro-inflammatory cytokines, and this is not double-stranded RNA.

Colorimetric heparinase assay for alternative anti-metastatic activity.

Heparanase has been previously associated with the metastatic potential, inflammation, and angiogenesis of tumor cells. Heparanase activity has been detected by means of UV absorption, radiolabeled substrates, electrophoretic migration, and heparan sulfate affinity assays. However, those methods have proven to be somewhat problematic with regards to application to actual biological samples, the accessibility of the immobilized substrates, experimental sensitivity, and the separation of degraded products. Rather than focusing on heparanase activity, then, we have developed a rapid, alternative colorimetric heparinase assay, on the basis of the recent finding that sulfated disaccharides generated from heparin by bacterial heparinase exhibit biological properties comparable to those from heparan sulfate by mammalian heparanase. In this study, the concentrations of porcine heparin and bacterial heparinase I were determined using a Sigma Diagnostics Kit. Morus alba was selected as a candidate through this assay system, and an inhibitor, resveratrol, was purified from its methanol extract. Its anti-metastatic effects on the pulmonary metastasis of murine B16 melanoma cells were also evaluated. Our findings suggest that this assay may prove useful as a diagnostic tool for heparinase inhibition, as an alternative anti-metastatic target.

Inhibition of PDGF-induced phospholipase C activation by herbimycin A.

Herbimycin A, an inhibitor of protein tyrosine kinases, dose-dependently reduced PDGF-induced inositol phosphates (IPt) accumulation without effect on phosphatidylethanol (PEt) formation in PLC-gamma 1-overexpressing NIH 3T3 (NIH 3T3 gamma 1) cells. The compound also reduced tyrosine phosphorylations of some proteins including PLC-gamma 1 in response to PDGF. On the other hand, phorbol 12-myristate 13-acetate (PMA)-induced phospholipase D (PLD) activation was reduced by herbimycin A in the cells, indicating that the pathways for PLD activation by PDGF and PMA are different from each other. Also, these results suggest that PLC-gamma 1 activation is not always an upstream event for PLD activation and that tyrosine phosphorylation of one or more proteins not affected by herbimycin A should be indispensable for PLD activation in PDGF-stimulated NIH 3T3 gamma 1 cells.

Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells.

An expression construct containing the cDNA encoding a modified aequorea green fluorescent protein (GFP) ligated to the 5'-end of the rat androgen receptor (AR) cDNA (GFP-AR) was used to study the intracellular dynamics of the receptor movement in living cells. In three different cell lines, ie. PC3, HeLa, and COS1, unliganded GFP-AR was seen mostly in the cytoplasm and rapidly (within 15-60 min) moved to the nuclear compartment after androgen treatment. Upon androgen withdrawal, the labeled AR migrated back to the cytoplasmic compartment and maintained its ability to reenter the nucleus on subsequent exposure to androgen. Under the condition of inhibited protein synthesis by cycloheximide (50 microg/ml), at least four rounds of receptor recycling after androgen treatment and withdrawal were recorded. Two nonandrogenic hormones, 17beta-estradiol and progesterone at higher concentrations (10(-7)/10(-6) M), were able to both transactivate the AR-responsive promoter and translocate the GFP-AR into the nucleus. Similarly, antiandrogenic ligands, cyproterone acetate and casodex, were also capable of translocating the cytoplasmic AR into the nucleus albeit at a slower rate than the androgen 5alpha-dihydrotestosterone (DHT). All AR ligands with transactivation potential, including the mixed agonist/antagonist cyproterone acetate, caused translocation of the GFP-AR into a subnuclear compartment indicated by its punctate intranuclear distribution. However, translocation caused by casodex, a pure antagonist, resulted in a homogeneous nuclear distribution. Subsequent exposure of the casodex-treated cell to DHT rapidly (15-30 min) altered the homogeneous to punctate distribution of the already translocated nuclear AR. When transported into the nucleus either by casodex or by DHT, GFP-AR was resistant to 2 M NaCl extraction, indicating that the homogeneously distributed AR is also associated with the nuclear matrix. Taken together, these results demonstrate that AR requires ligand activation for its nuclear translocation where occupancy by only agonists and partial agonists can direct it to a potentially functional subnuclear location and that one receptor molecule can undertake multiple rounds of hormonal signaling; this indicates that ligand dissociation/inactivation rather than receptor degradation may play a critical role in terminating hormone action.

Quinidine blockade of the carbachol-activated nonselective cationic current in guinea-pig gastric myocytes.

1. In guinea-pig gastric myocytes isolated from the antral circular layer, stimulation of muscarinic receptors by carbachol (CCh) induces a cationic current (ICCh) which is known as the main mechanism of depolarization induced by muscarinic stimulation. 2. We tested the effects of a number of ion channel blockers on ICCh and focused upon quinidine which was a highly potent blocker. Externally applied quinidine suppressed ICCh (IC50 = 0.25 microM) in a reversible and voltage-dependent manner. Applied internally, quinidine was about 100 times less potent than when applied externally. Persistent activation of G-protein by GTP gamma S also induced a cationic current similar to ICCh and this current was also blocked by quinidine. 4-Aminopyridine and tetraethylammonium also suppressed ICCh in a dose-dependent manner (IC50 = 3.3 mM and 4.1 mM, respectively). 3. Pretreatment with quinidine (2 microM) selectively blocked the acetylcholine (ACh)-induced depolarization which was recorded in the multicellular tissues by a conventional intracellular microelectrode technique. 4. Voltage-dependent K-currents were also suppressed by quinidine but in a higher concentration range (IC50 = 3 microM). Quinidine, 10 microM, decreased the amplitude of the voltage-dependent Ca current to only a small extent (15% decrease at 0 mV). Quinidine, 2 microM, also suppressed only a minute proportion of the Ca-activated K current (11.1% decrease at 45 mV). 5. From these experiments, it is concluded that some organic agents known as K channel blockers are able to block the CCh-activated cation channel in a non-specific manner and among them, quinidine can be used as an effective blocker for ICCh in guinea-pig gastric myocytes.

Androgen receptor: structural domains and functional dynamics after ligand-receptor interaction.

Androgens are C-19 steroids secreted primarily from the testes and adrenals that play a critical role in reproduction. Reproductive functions of androgens are mediated through coordination of diverse physiological processes ranging from brain functions to specific cell proliferation and apoptosis. At the molecular level, most of these regulatory influences are exerted by altered expression of appropriate genes by the androgen receptor (AR), a member of the nuclear receptor (NR) superfamily. The unliganded AR is a cytoplasmic protein and, upon ligand binding, it translocates into the nucleus. Thereafter, in conjunction with other transcription factors and coactivators, the AR influences transcription of target genes through a multistep process that includes its clustering in a subnuclear compartment. Here, we describe the genomic organization of the AR, the role of individual structural domains in specific AR function, and the influence of agonistic/antagonistic ligands in the intracellular movement of the receptor. We also show that the AR is capable of undergoing multiple rounds of nucleocytoplasmic recycling after ligand binding and dissociation. Xenobiotic ligands, considered as selective androgen receptor modulators (SARMs), can modulate AR activity by inhibiting either its nuclear translocation or its subnuclear clustering and subsequent transactivation function.

Effects of purinergic agonists on mechanical and electrical activities of gastric smooth muscle of guinea-pig.

The effects of purinergic agonists on the mechanical and electrical activity of antral circular muscle of guinea-pig stomach were investigated. ATP potentiated the mechanical contraction without any change in membrane potential. To characterize the purinoceptors involved, effects of ATP analogues, especially P2X or P2Y receptor agonists, were studied. 2-MethylthioATP (2-MeSATP), a P2Y agonist, showed the same effects as that of ATP. On the other hand, a P2X agonists, alpha, beta-methyleneATP (alpha, beta-MeATP) incuced relaxation with hyperpolarization. At the holding potential of 0 mV, spontaneous transient outward currents (STOCs) were recorded in whole-cell mode. Externally applied ATP revealed transient increase of STOCs. Considering that STOCs reflect the changes of internal Ca2+, these results might be explained by the transient increase of internal Ca2+ release from internal Ca2+ store. From these results, it is concluded that there are at least two types of purinoceptors in guinea-pig stomach, and contractile response via P2Y-purinoceptor might be related to the elevation of internal Ca2+ released from internal Ca2+ store.

Effect of intracellular cAMP on K current in single gastric myocytes of guinea-pig.

In guinea-pig stomach, the effect of increased intracellular cAMP induced by forskolin, 8-Br-cAMP, or isobuthyl-methylxanthine (IBMX) on K current was studied in whole-cell currents. In enzymatically dispersed single cells, agents elevating [cAMP]in showed quite different effects on the IK depending on the recording conditions. Though using the same recording pipette containing 0.1 mM EGTA, agents elevating [cAMP]in reduced IK(Ca) recorded at various membrane potentials, while forskolin increased the spontaneous transient outward currents (STOCs) recorded at fixed holding potential, 0 mV. Such a reduction of K currents was not observed in the cells dialysed with heparin contained in the recording pipette. These results suggested the possibility that the cAMP modulate the internal Ca(2+)-store. To assess the effect of cAMP on the internal Ca2+ store, carbachol or caffeine was used to mobilize intracellular Ca2+ store. Using the carbachol- or caffeine-induced IK(Ca), the effects of forskolin on [Ca2+]in was investigated. Forskolin inhibited the carbachol-induced K current, while the caffeine-induced current was not affected. From the above results, it might be suggested that increased intracellular cAMP might modulate the release of Ca2+ from IP3-sensitive Ca2+ store to- reduce the Ca(2+)-activated K currents.

Dual roles of phorbol 12, 13-dibutyrate in the regulation of guinea-pig gastric contraction.

We performed experiments to investigate the actions of protein kinase C(PKC) on mechanical contraction during agonist stimulation in guinea pig stomach. We used carbachol and high K condition to enhance mechanical contraction by mobilizing intracellular Ca2+ and increasing Ca2+ influx through voltage-dependent Ca(2+)-channel, respectively. Phorbol 12, 13-dibutyrate (PDBu) increased spontaneous contractions sensitive to verapamil (438 +/- 82.2%, n = 7) and potentiated high K-induced contraction (189 +/- 22.5%, n = 5). However, carbachol (CCh)-induced contractions in PDBu-treated condition depended on extracellular Ca2+. In the presence of extracellular Ca2+, CCh-induced contraction was potentiated, while it was suppressed in the absence of extracellular Ca(2+)-preloaded muscle strips. To prove the hypothesis that such phenomena might be related with changes of myoplasmic Ca2+ concentration, we investigated the effect of PDBu on voltage-dependent Ca2+ current(ICa) and CCh-induced Ca2+ activated K current(IK(Ca)) transient using whole-cell voltage clamp technique. For recording voltage-dependent Ca2+ current(ICa), 10 mM Ba2+, instead of Ca2+, was used to enhance the current size. Voltage-dependent Ba2+ current(IBa) was increased by PDBu (212 +/- 32.2% of steady state currents, n = 5), while CCh-induced increase of IK(Ca) transient was inhibited by PDBu (n = 5), the changes of which were similar to those of muscle contractions. To analyze the steps involved in the inhibition of CCh-induced IK(Ca) transient by PDBu, we investigated the effect of PDBu on IK(Ca) in the cells perfused with Ins (1, 4, 5)P3. However, Ins (1, 4, 5)P3 induced IK(Ca) was not inhibited by the treatment with phorbol ester. From these results, it is concluded that inhibition of phosphatidylinositol phospholipase C(PI-PLC) system and potentiation of ICa by PKC are important regulatory mechanisms in agonist-induced muscle contraction.

Behavioral effects and binding affinities of the fentanyl derivative OHM3507.

Several fentanyl derivatives have been reported to have novel pharmacologies that might be exploited for developing alternate approaches to the treatment of pain. The purpose of the current series of studies was to evaluate OHM3507, a novel fentanyl derivative reported to have an unusual pharmacological profile in nonprimate species. Similar to several other fentanyl derivatives with clinical potential, OHM3507 had the highest affinity (IC50 = 10 nM) for mu ([3H]D-Ala2,N-Me-Phe4,Gly5-OH-labeled) receptors with 6- and 176-fold lower affinity for delta ([3H]D-Pen2-D-Pen5-labeled), and kappa ([3H]ethylketocyclazocine-labeled) receptors, respectively. In rhesus monkeys, OHM3507 shared discriminative stimulus effects with morphine, increased tail-withdrawal latencies in a warm-water procedure of antinociception, decreased ventilation in monkeys breathing normal air or 5% CO2, and failed to modify accuracy on acquisition and performance tasks up to doses that decreased rates of food-maintained responding. The opioid antagonists naltrexone and naltrindole antagonized the behavioral effects of OHM3507 in a manner that was consistent with mu-receptor mediation. Although OHM3507 appeared to have low efficacy opioid actions in nonprimate species, results from the current studies clearly show this compound to have strong, fentanyl-like mu agonist actions in rhesus monkeys. These results provide another example of the sometimes poor predictability in the behavioral pharmacology of fentanyl derivatives among species, in this case between monkeys and rats, mice and rabbits, and demonstrates the need for evaluating new drugs under a broad range of conditions to increase the probability of identifying novel compounds that can be used to treat pain.

Topical anti-inflammatory activity of dianemycin isolated fromStreptomyces sp. MT 2705-4.

In order to develop new anti-inflammatory agents having different action mechanisms compared with nonsteroidal and steroidal anti-inflammatory drugs, the culture broths of various actinomycetes isolated from soil were screened using anin vivo mouse ear edma assay and one strain (Streptomyces sp. MT 2705-4: KCTC 8651P) was selected. Activity-guided purification led to the isolation of a polyether compound, dianemycin. Topically, dianemycin showed a potent anti-inflammatory activity in mouse ear edema induced by croton-oil or arachidonic acid. ED(50) value of dianemycin was found to be 0.8 mg/ear compared to 0.4 mg/ear of prednisolone in croton-oil ear edema. However, dianemycin did not show the inhibitory activity in UV-erythema and delayed hypersensitivity reaction. These results indicate that dianemycin is a potential topical anti-inflammatory agent.

Short-term vestibular responses to repeated rotations.

To investigate the short-term vestibular habituation, we performed the 4 successive velocity step tests on 28 volunteers, the peak velocity of which was 100 deg/sec with acceleration and deceleration of 100 deg/sec2. As the repeated rotations might alter the vestibulo-ocular reflex (VOR), sinusoidal rotations at a frequency of 0.16 Hz were also given before and after the 4 successive velocity step tests to investigate the changes of gain and phase of VOR. The 28 volunteers were divided into two groups, group I and II, according to their responses to repeated rotations. In group I (25 subjects, mean age 23.7 year), the mean values of the slow cumulative eye position (SCEP), the time integral of eye velocity during nystagmus, was reduced after each trial of the 4-successive velocity step tests as follows; 403.4 +/- 29 degree (1st trial), 346.2 +/- 37 degree (2nd trial), 278.3 +/- 33 degree (3rd trial) and 256.6 +/- 36 degree (4th trial). The time constant of the nystagmus was also reduced as follows; 12.9 +/- 0.78 second (1st trial), 12 +/- 0.63 second (2nd trial), 9.7 +/- 0.78 second (3rd trial) and 9.9 +/- 0.54 second (4th trial). In group II (3 subjects, mean age 28.3 year), the mean values of SCEP gradually increased; 774.3 +/- 135 degree (1st trial), 1127 +/- 178 degree (2nd trial), 1096.3 +/- 123.4 degree (3rd trial) and 1225.7 +/- 199.7 degree (4th trial). The time constant of the nystagmus increased; 15.7 +/- 2.7 second (1st trial), 22 +/- 4.5 second (2nd trial), 22.3 +/- 3.3 second (3rd trial) and 23.3 +/- 5.7 second (4th trial). The gain of vestibulo-ocular reflex (VOR) induced by sinusoidal rotations at a frequency 0.16 Hz increased in both groups: 0.59 +/- 0.03 to 0.78 +/- 0.06 (group I) and 0.65 +/- 0.07 to 1.15 +/- 0.06 (group II). Phase changes were also observed. In group I, the phase was shifted from 0.6 +/- 0.6 degree to -0.40 +/- 0.6 degree. In group II, the phase was shifted from 3.67 +/- 1.86 degree to -0.33 +/- 0.33 degree. The repeated rotation did not induce a common nystagmic response in all subjects. Thus, person to person variations should be considered in short term vestibular habituation.

Clinical assessment of adventitious movements.

Many procedures with variable validity and reliability have been developed in research settings to evaluate adventitious movements and related phenomena in specific populations, e.g., people with schizophrenia treated with dopamine antagonists, but these only provide global assessments or rate specific movements. A battery for rating individuals with possible movements disorders in a comprehensive way in clinical settings is needed so a protocol to assess briefly and thoroughly potential movement disorders was videotaped for five prepubertal boys with autistic disorder and severe mental retardation in a clinical trial. Utilizing a Movement Assessment Battery, four raters independently scored videotapes of 10-16 movements assessments of each of the five subjects. Experienced raters attained agreement of 59% to 100% on ratings of tardive dyskinesia and 48% to 100% on tics. Hindrances to reliability included poor quality of some tapes, high activity of subjects, and fatigue of raters.

A review of the literature and a preliminary study of family compliance in a developmental disabilities clinic.

To investigate the compliance of family members with the treatment recommended for patients, three child and adolescent psychiatrists assessed the charts of all active outpatients in a developmental disabilities clinic in the psychiatric department of a tertiary care municipal hospital utilizing a Family Compliance Checklist, a survey form for chart review, in October, 1993 (n = 40), and in April, 1994 (n = 41). Almost no clients missed appointments over a 6-mo. period. Only one family refused to permit the use of medication. Three families refused to make appointments. The majority of the patients were Hispanic and almost half were Roman Catholic. We conclude that most families of patients in a developmental disabilities clinic comply with recommended treatment plans including scheduled appointments and prescribed medications.

Clinical assessment of self-injurious behavior.

The Timed Self-injurious Behavior Scale is an observational scale rating the frequency of 16 types of self-injurious behaviors during each 10-sec. interval of a 10-min. observation period. Advantages of the scale are utilization of direct observation and independence from the variable recollection of symptoms by subjects and care givers. 19 videotaped sessions of a subject who exhibited eight types of self-injurious behaviors were rated with the scale independently by three raters. Eighty percent and better agreement was found for the four specific forms of those behaviors exhibited by the subject sufficiently frequently, self biting, head punching, head slapping, and hair removal.

Dyskinesias subside off all medication in a boy with autistic disorder and severe mental retardation.

A boy with autistic disorder and severe mental retardation developed severe dyskinesias, including objective akathisia (probable) and tics, a month after discontinuation of at least two years of treatment with drugs block dopamine receptors. These dyskinesias greatly subsided during a 17-wk. open-label nonblind clinical trial of clomipramine, and returned transiently when the parents abruptly discontinued clomipramine. However, the dyskinesias gradually subsided during two and a half years of follow-up with the boy being off all medication. A few stereotypies remain. We believe this suggests the hypothesis that movement disorders, such as withdrawal and tardive akathisia and tics, occurring in boys with autistic disorder treated with dopamine receptor-blocking drugs may subside months or years after discontinuation of the agents and that clomipramine may facilitate this process. We also hypothesize that some boys with autistic disorder and mental retardation exhibit fewer movement disorders, fewer psychiatric symptoms, and better over-all functioning after they have received no dopamine receptor-blocking drugs for several months, and this improvement continues years after the medication has ceased.

Cellular distributions of the prohormone processing enzymes PC1 and PC2.

The prohormone convertases PC1 (also known as sPC3) and PC2 are known to mediate the proteolytic conversion of inactive neuropeptide and hormone precursors to bioactive peptide products. In this study we have used sucrose density centrifugation to determine the subcellular distributions of the various forms of PC1 and PC2 in three different cell types, AtT-20, beta TC3, and PC12 cells. The former two cell lines naturally express PC enzymes, while PC12 cell clones expressing PCs were obtained by stable transfection. Our data show considerable cell-line specific variation in PC processing, with PC12 cells exhibiting the most complete processing of both enzyme precursors. While in all cell lines mature forms of both enzymes were stored within particles having the same buoyant density as secretory granule markers, in some cell lines substantial amounts of mature PC1 and PC2 were also associated with the Golgi marker. Processing of the two PC precursors was not interdependent since PC12 cells expressing only one of the two PCs were fully capable of enzyme maturation. Interestingly, analysis of intracellular processing of an endogenous peptide precursor, proneurotensin, revealed that transfected PC1, but not PC2, showed enzymatic activity against this precursor.

Role of calmodulin in the activation of carbachol-activated cationic current in guinea-pig gastric antral myocytes.

In mammalian gastrointestinal myocytes, it is known that muscarinic stimulation activates nonselective cation channels through a G-protein and a Ca2+-dependent pathway. We recorded inward cationic currents following application of carbachol (ICCh) to guinea-pig gastric myocytes, which were held at -20 mV using the whole-cell patch-clamp method. ICCh was suppressed by nicardipine or removal of Ca2+ from the bath solution. The peak value of inward current induced by repetitive applications of carbachol (CCh) decreased progressively (run-down phenomenon). This run-down was significantly alleviated by the addition of calmodulin to the pipette solution (0.15 mg/ml) or by using the perforated-patch whole-cell voltage-clamp technique. Moreover, W-7[N-6(aminohexyl)-5-chloro-1-naphthalenesulphonamide], a calmodulin antagonist, was a reversible inhibitor of ICCh. However, @-7 had only a weak inhibitory effect on the same cationic current which was induced by guanosine 5'-O-(3-thiotriphosphate) (GTP¿gammaS] 0.2 mM) in the pipette solution. This GTP[gammaS]-induced cationic current was still markedly suppressed by the Ca2+-free bath solution. W-7 itself had a weak inhibitory effect on voltage-operated Ca2+ channels as well as the effects on ICCh. These data suggest that multiple Ca2+-dependent pathways are involved in the activation of CCh-gated cation channels in guinea-pig antral myocytes and a Ca2+/calmodulin-dependent pathway would be one of them.