Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1.

Two seven-gene phenazine biosynthetic loci were cloned from Pseudomonas aeruginosa PAO1. The operons, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, are homologous to previously studied phenazine biosynthetic operons from Pseudomonas fluorescens and Pseudomonas aureofaciens. Functional studies of phenazine-nonproducing strains of fluorescent pseudomonads indicated that each of the biosynthetic operons from P. aeruginosa is sufficient for production of a single compound, phenazine-1-carboxylic acid (PCA). Subsequent conversion of PCA to pyocyanin is mediated in P. aeruginosa by two novel phenazine-modifying genes, phzM and phzS, which encode putative phenazine-specific methyltransferase and flavin-containing monooxygenase, respectively. Expression of phzS alone in Escherichia coli or in enzymes, pyocyanin-nonproducing P. fluorescens resulted in conversion of PCA to 1-hydroxyphenazine. P. aeruginosa with insertionally inactivated phzM or phzS developed pyocyanin-deficient phenotypes. A third phenazine-modifying gene, phzH, which has a homologue in Pseudomonas chlororaphis, also was identified and was shown to control synthesis of phenazine-1-carboxamide from PCA in P. aeruginosa PAO1. Our results suggest that there is a complex pyocyanin biosynthetic pathway in P. aeruginosa consisting of two core loci responsible for synthesis of PCA and three additional genes encoding unique enzymes involved in the conversion of PCA to pyocyanin, 1-hydroxyphenazine, and phenazine-1-carboxamide.

phzO, a gene for biosynthesis of 2-hydroxylated phenazine compounds in Pseudomonas aureofaciens 30-84.

Certain strains of root-colonizing fluorescent Pseudomonas spp. produce phenazines, a class of antifungal metabolites that can provide protection against various soilborne root pathogens. Despite the fact that the phenazine biosynthetic locus is highly conserved among fluorescent Pseudomonas spp., individual strains differ in the range of phenazine compounds they produce. This study focuses on the ability of Pseudomonas aureofaciens 30-84 to produce 2-hydroxyphenazine-1-carboxylic acid (2-OH-PCA) and 2-hydroxyphenazine from the common phenazine metabolite phenazine-1-carboxylic acid (PCA). P. aureofaciens 30-84 contains a novel gene located downstream from the core phenazine operon that encodes a 55-kDa aromatic monooxygenase responsible for the hydroxylation of PCA to produce 2-OH-PCA. Knowledge of the genes responsible for phenazine product specificity could ultimately reveal ways to manipulate organisms to produce multiple phenazines or novel phenazines not previously described.

The effect of flicker rate on measured visual field extent in very young children.

PURPOSE: To investigate the effect of flicker rate on measured visual field extent in toddlers. METHODS: A total of 270 full-term children (90 each at 11-, 17-, and 30-months of age) and 36 adults were tested binocularly with an LED static perimetry procedure using a black double-arc perimeter. Each subject was tested with one of three flicker rates: 0, 3, or 10 Hz. The median farthest location seen and an interpolated estimate of the location at which 50% of the subjects detected the peripheral stimulus were calculated for each age group for each flicker rate. RESULTS: For 11-, 17-, and 30-month-old subjects, but not adults, flickering stimuli produced a larger measured visual field extent than nonflickering stimuli. For the 10-Hz stimuli, measured visual field extent in children did not differ from that of adults. CONCLUSIONS: In infants and young children, binocular measured visual field extent is enhanced by peripheral stimulus flicker. Maturity of the measured visual field depends on the stimulus parameters used during testing.

A molecularly identified P2Y receptor simultaneously activates phospholipase C and inhibits adenylyl cyclase and is nonselectively activated by all nucleoside triphosphates.

We recently cloned and expressed a novel P2Y receptor (tp2y receptor) from a turkey cDNA library. Expression of this receptor in 1321N1 human astrocytoma cells confers nucleotide-dependent stimulation of phospholipase C activity; however, as we demonstrate here, it also confers nucleotide-dependent inhibition of adenylyl cyclase. Both the phospholipase C and adenylyl cyclase responses were promoted by receptor agonists over a similar range of concentrations. Moreover, not only did UTP and ATP activate the avian receptor but ITP, GTP, xanthosine 5'-triphosphate, and CTP were also agonists, with EC(50) values ranging between 0.1 and 1 microM. Similar potencies, rank-order, and selectivity of nucleotide agonists were also demonstrated for intracellular Ca(2+) mobilization measured during a 30-s stimulation under constant superfusion conditions. This observation indicates that receptor activation by nucleoside 5'-triphosphates is not produced by interconversion of these nucleotides into ATP or UTP. Pretreatment of cells with pertussis toxin completely abolished the inhibitory effect of nucleotide agonists on adenylyl cyclase, whereas the activation of phospholipase C was only partially inhibited. These results demonstrate that the avian P2Y receptor is a nucleoside triphosphate receptor of broad agonist selectivity that interacts with both pertussis toxin-insensitive and -sensitive G proteins to activate phospholipase C and to inhibit adenylyl cyclase. This is the first cloned P2Y receptor that is clearly Gi/adenylyl cyclase-linked.

Stimulus motion increases measured visual field extent in children 3.5 to 30 months of age.

PURPOSE: To examine the influence of stimulus motion on measured visual field extent of 3.5- to 30-month-old children and adults. METHODS: Each subject was tested with LED-hybrid and LED-kinetic perimetry procedures, using a black double-arc perimeter. Targets in both procedures were identical in size, color, luminance, contrast, and flicker rate. However, in the LED-hybrid procedure, peripheral targets were sequentially illuminated from more peripheral to more central locations, whereas in the LED-kinetic procedure, a peripheral target on a black wand was manually moved centrally along the perimeter arm. A subset of subjects was also tested with white sphere kinetic perimetry (WSKP). RESULTS: The LED-kinetic procedure produced larger measured visual field extent than the LED-hybrid procedure in 3.5-, 11-, 17-, and 30-month-olds, but not in 7-month-olds or adults. Data from subjects tested with WSKP indicated that both stimulus motion and discrepancies in scoring methods contributed to the difference reported previously between visual field measurements obtained with WSKP vs. LED-hybrid perimetry. CONCLUSION: In infants and toddlers, measured visual field extent is larger for moving than for nonmoving targets. Further research is needed to determine whether the effect of motion is related to the visual system or to attentional factors.

Does rate of stimulus presentation affect measured visual field extent in infants and toddlers?

PURPOSE: To evaluate the effect of stimulus presentation rate on the measurement of visual field extent in infants and toddlers. METHODS: Visual field extent was measured for 300 children (N = 60 at 3.5, 7, 11, 17, and 30 months) and 24 adults using hybrid static-kinetic perimetry. Flickering light-emitting diode (LED) stimuli were illuminated sequentially, peripherally to centrally at 10.2 degrees intervals, along 4 diagonal meridia at 2 stimulus presentation rates: 2 s/stimulus (equivalent to 5 degrees/s) and 3 s/stimulus (equivalent to 3 degrees/s). Rate of presentation was a between-subjects variable. RESULTS: No effect of stimulus presentation rate was found for adults. The faster rate of stimulus presentation yielded smaller measured visual field extent for children between the ages of 7 and 30 months. The apparent difference seen with 3.5-month-olds did not reach significance. CONCLUSIONS: Faster rates of stimulus presentation may result in underestimation of visual field extent in children between the ages of 7 and 30 months.

Levels of endogenous adenosine in rat striatum. I. Regulation by ionotropic glutamate receptors, nitric oxide and free radicals.

Glutamate release after ischemia, hypoxia and seizure activity plays an important role in stimulating adenosine production and release. We characterized the ionotropic glutamate receptor subtype that regulates adenosine levels in vivo and investigated the role of nitric oxide and free radicals in mediating N-methyl-D-aspartate (NMDA)-induced increases in adenosine levels. Rats received unilateral intrastriatal injections and were sacrificed 15 min postinjection by high-energy focused microwave irradiation (10 kW, 1.25 s). Adenosine levels were measured by high-performance liquid chromatography in ipsilateral and contralateral striata. NMDA and kainic acid dose-dependently increased levels of adenosine whereas (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazol proprionic acid had no effect. The NMDA- and kainic acid-induced increases were blocked by dizocilpine, and the kainic acid response was decreased by 6-cyano-7-nitroquinoxaline-2,3-dione. The effects of NMDA and kainic acid on levels of adenosine were not additive. Intrastriatal L-arginine decreased, and the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester, increased basal adenosine levels. Coadministration of NMDA with L-arginine or NG-nitro-L-arginine methyl ester did not significantly affect NMDA-induced increases in levels of adenosine. N-Tert-butyl-phenylnitrone, a free radical scavenger, reversed L-arginine-induced decreases and NMDA-induced increases in levels of adenosine. Together, these results indicate that NMDA-type ionotropic receptors play an important role in regulating in vivo levels of adenosine in rat striatum and that free radicals, but not nitric oxide, apparently are involved in NMDA-induced increases in levels of adenosine. Conversely, nitric oxide, but not free radicals, apparently exert tonic control over basal levels of endogenous adenosine.

Levels of endogenous adenosine in rat striatum. II. Regulation of basal and N-methyl-D-aspartate-induced levels by inhibitors of adenosine transport and metabolism.

Selective inhibitors of adenosine production, degradation and transport were used to potentiate in vivo levels of adenosine and to determine the source of both basal and N-methyl-D-aspartate (NMDA)-induced increases in levels of endogenous adenosine in vivo. Male Sprague-Dawley rats receiving unilateral intrastriatal injections of pharmacological agents were sacrificed 15 min postinjection by high-energy focused microwave irradiation (10 kW, 1.25 s). Ipsilateral and contralateral striata were dissected, and adenosine levels were measured by high-performance liquid chromatography. Inhibition of 5'-nucleotidase by alpha, beta-methylene ADP dose-dependently decreased adenosine levels under basal as well as NMDA-stimulated conditions. Inhibition of nucleoside transport by dilazep and adenosine deaminase by 2'-deoxycoformycin each dose-dependently increased basal adenosine levels. 2'-Deoxycoformycin potentiated NMDA-induced increases in adenosine levels. Inhibition of adenosine kinase by 5'-amino-5'-deoxyadenosine increased basal levels of adenosine, but did not significantly affect NMDA-induced increases in adenosine. 2'-Deoxycoformycin combined with 5'-amino-5'-deoxyadenosine produced a greater enhancement of NMDA-induced increases in levels of adenosine than when either drug was administered separately. Endogenous adenosine in vivo apparently originates from release of adenosine as well as from release and extracellular breakdown of a nucleotide under both basal and NMDA-stimulated conditions. Furthermore, inhibitors of adenosine kinase and adenosine deaminase work best to increase levels of endogenous adenosine under basal and NMDA-stimulated conditions, respectively.

Diadenosine polyphosphates inhibit adenosine kinase activity but decrease levels of endogenous adenosine in rat brain.

Findings in peripheral tissues that diadenosine polyphosphates (Ap(n)As) activate 5'-nucleotidase activity and inhibit adenosine kinase activity in vitro led us to test the hypothesis that Ap(n)As and analogues thereof, through such actions on purine enzymes, increase brain levels of endogenous adenosine in vivo. Accordingly, we tested Ap(n)As for their effects on the in vitro activities of adenosine kinase, adenosine deaminase, AMP deaminase and 5'-nucleotidase and, following unilateral microinjections in rat striatum, on in vivo levels of endogenous adenosine. Adenosine kinase activity was not affected significantly by 5',5'''-P1,P2-diadenosine pyrophosphate (Ap2A) or by 5',5'''-P1,P3-diadenosine triphosphate (Ap3A), but was inhibited by 5',5'''-P1,P4-diadenosine tetraphosphate (Ap4A), 5',5'''-P1,P5-diadenosine pentaphosphate (Ap5A) and 5',5'''-P1,P6-diadenosine hexaphosphate (Ap6A); apparent IC50 values were 5.0, 3.3 and 500 microM, respectively. Inhibition of adenosine kinase activity by Ap4A and the four metabolically stable analogues of Ap4A tested was uncompetitive. Following unilateral intrastriatal injections, adenosine levels, relative to uninjected contralateral striatum, were decreased significantly (P < 0.05) by 48% with Ap4A and by 37% with AppCH2ppA, a metabolically stable analogue of Ap4A. Striatal levels of adenosine were not affected significantly by Ap5A or Ap6A. Cytosolic, but not particulate 5'-nucleotidase activity was inhibited and AMP deaminase activity was increased by some Ap(n)As. Although adenosine kinase inhibitors increase levels of endogenous adenosine and we showed here that Ap(n)As were potent inhibitors of this enzyme, these particular actions of Ap(n)As were not consistent with their effects on levels of endogenous adenosine.

Brain regional levels of adenosine and adenosine nucleotides in rats killed by high-energy focused microwave irradiation.

A high-energy focused microwave system for killing experimental animals was used to rapidly inactivate enzymes and prevent postmortem breakdown of adenine nucleotides and adenosine, thereby enabling accurate measurements of AMP, ADP, ATP and adenosine in rat brain. For comparison, purine levels were measured in brains of rats killed by decapitation, decapitation into liquid nitrogen, or in situ freezing of the brain with liquid nitrogen. Of the three microwave irradiation power levels used, 10, 6.0 or 3.5 kW, rats killed by 10 kW had the highest ATP levels (28.8 nmol/mg protein) and cellular energy charge value (0.8), and the lowest levels of AMP (2.2 nmol/mg protein) and adenosine (19.7 pmol/mg protein). Of the 6 brain regions studied, adenosine levels (pmol/mg protein) ranged from 10 in cerebral cortex to 170 in cerebellum of rats killed using 10 kW microwave irradiation and, for comparison, ranged from 840 in cerebral cortex to 2498 in striatum of rats killed by decapitation. Focused microwave killing permits precise and accurate measurements of purines in discrete regions of rat brain.

Enhancement of NMDA-induced increases in levels of endogenous adenosine by adenosine deaminase and adenosine transport inhibition in rat striatum.

Unilateral microinjection of N-methyl-D-aspartate (NMDA) into striatum of rats subsequently killed by high-energy focused microwave irradiation significantly increased in vivo levels of endogenous adenosine. At a dose of 25 nmol NMDA, levels of adenosine in injected striata were 263% of levels in uninjected contralateral striata. An inhibitor of adenosine deaminase (deoxycoformycin, DCF) in combination with an inhibitor of adenosine transport (dilazep, DLZP) at a dose that did not affect levels of endogenous adenosine, potentiated NMDA-induced increases in adenosine levels to 426% of contralateral striata. In the presence of DCF and DLZP, NMDA dose-dependently increased levels of adenosine (% of contralateral striatum) from 166% at 10 nmol to 622% at 100 nmol. NMDA-induced increases in levels of endogenous adenosine were completely blocked by prior administration of the NMDA receptor antagonist MK 801 (dizocilpine). Inhibitors of adenosine metabolism and transport may provide therapeutic benefit by potentiating excitatory amino acid-induced increases in levels of endogenous adenosine in vivo.

[3H]paroxetine binding to platelets of patients with social phobia: comparison to patients with panic disorder and healthy volunteers.

Recent studies suggest that serotonergic functioning may be aberrant in patients with social phobia. Capacity of the serotonin (5-HT) transporter, as determined by 3H-paroxetine binding, was measured in 18 drug-free patients with generalized social phobia and compared to 15 drug-free patients with panic disorder and 23 healthy control subjects. The density (Bmax) and affinity (1/Kd) of 3H-paroxetine binding sites was similar in all three groups. To the extent that the serotonin transporter in platelets and neurons is comparable, these findings suggest that this aspect of serotonergic function is normal in patients with social phobia.

Chronic changes in thyroid hormones do not affect brain adenosine receptors.

1. In this study, the authors examined the effects of chronic (14 days) changes in thyroid function on a major neuromodulatory receptor system in the brain- the adenosinergic system. While previous investigators have examined the effects of alteration in thyroid function on adenosine receptors in peripheral tissues (adipocytes), this is the first study to examine such effects in brain. 2. Three groups of male Sprague-Dawley rats were treated for 14 days with either a) oral PTU (0.00625%), iodine-free diet, and i.p. saline injections, b) i.p. saline injections, or c) i.p. triiodothyronine (25 micrograms/100 g) injections. 3. These manipulations reliably resulted in the production of hypothyroidism (TSH 30.2 +/- 8.6 ng/ml), euthyroidism (TSH 2.1 +/- 0.9), and hyperthyroidism (TSH < 0.4). 4. Treatment had no significant effect on the Bmax or Kd of [3H]DPCPX (A1-antagonist) binding to homogenates from cerebral cortex, cerebellum or hippocampus; similarly, no effect on [3H]CGS-21680 (A2-agonist) binding to striatal homogenates was noted. 5. Similarly, quantitative autoradiographic studies failed to reveal consistent regional alterations unique to either hypo- or hyperthyroidism. 6. Incubation of sections with GppNHp resulted in the expected reduction (approximately 40%) in agonist binding, but there was no differential effect seen for either the hypo- or hyperthyroid tissues. 7. These preliminary findings suggest that alterations in brain adenosine receptors or G-protein-receptor coupling are unlikely to be requisite correlates of abnormal thyroid hormone levels.

Lymphocyte beta-adrenoceptors in social phobia.

Beta-adrenergic receptor kinetics were measured in leukocytes from 17 drug-free, nondepressed patients with social phobia (generalized type) and 17 gender-matched and age-matched healthy controls. Binding was characterized using the highly specific beta-adrenergic ligand [125I]pindolol (125IPIN). Contrary to some studies in panic disorder and many studies in depression, no significant difference was found in Bmax or Kd values between social phobic patients and controls. Neither severity of social phobic symptoms nor the severity of certain symptoms of beta-adrenergic activation (i.e., tachycardia, tremor, blushing) influenced Bmax or Kd. To the extent that these peripheral indices can be considered reflective of central processes, these findings suggest that a simple defect in beta-adrenoceptor number of affinity is unlikely to explain the pathophysiology of generalized social phobia.

Failure of 2'-deoxycoformycin to protect against transient forebrain ischemia in rat.

We determined whether 2'-deoxycoformycin (DCF), a potent highly specific inhibitor of adenosine deaminase (ADA), protected against transient forebrain ischemic neuronal injury in rat. Anesthetized male Sprague-Dawley rats received i.p. injections of either saline, 0.5 mg/kg or 5 mg/kg DCF 2 h before undergoing a 10-min forebrain ischemic insult induced by bilateral carotid artery occlusion with concomitant hypotension. Rat brain sections taken 7 days post-ischemia showed damage mostly in the CA1 region of the hippocampus. Quantification of neuronal injury showed no significant differences between saline- or DCF-treated rats. These results indicate that, contrary to previous reports, DCF does not protect against the neuronal damage that follows forebrain ischemia in rat.

An evaluation of therapeutic programs for the treatment of secondary inorgasmia in women.

This paper is concerned with a review and evaluation of programs for the treatment of secondary inorgasmia in women. Treatments address a wide range of factors. They include medical and psychiatric disorders, lack of sexual knowledge and communication between partners, marital disharmony, sexual anxiety, and performance anxiety. These treatment approaches are evaluated, along with the effectiveness of Masters and Johnson's therapy, and the many variants of this approach. The use of erotic fantasy in the treatment of this condition is also discussed. Conclusions are limited by the nature of research in this area. It is essentially clinical in form. Often it fails to define the characteristics of the conditions that are required to assess contributing factors, treatment strategies, and pre- and posttreatment measures.

Rat brain adenosine deaminase after 2'-deoxycoformycin administration: biochemical properties and evidence for reduced enzyme levels detected by 2'-[3H]deoxycoformycin ligand binding.

Near total inhibition of brain adenosine deaminase (ADA) activity in rats injected with the potent ADA inhibitor 2'-deoxycoformycin (DCF) was previously shown to reduce enzyme activity for up to 50 days during which time the enzyme exhibited reduced sensitivity to in vivo inhibition by DCF. Here, we investigated the biochemical properties of ADA and the basis for its reduced activity after DCF treatment. It was found that much higher doses of DCF were required to inhibit ADA in DCF-treated compared with drug-naive animals. Fourteen days after DCF administration, reduced ADA activity in brain homogenates was due to a decrease in Vmax, rather than to an altered Km of ADA for adenosine. DCF treatment had no effect on Ki values for erythro-9-(2-hydroxy-3-nonyl)adenine inhibition of ADA. The IC50 value for DCF inhibition of ADA in hypothalamus was unchanged. However, the Ki for DCF inhibition of ADA in whole brain increased by fivefold. Sucrose gradient analysis of brain ADA revealed only one corresponding peak of activity and [3H]DCF-labeled ADA in DCF-treated and control rats. A radioligand filtration assay with [3H]DCF was developed to assess the effects of DCF on ADA protein levels. Over a roughly 200-fold range of ADA activities the binding of [3H]DCF was highly correlated with deaminase activity (r = 0.99). In brain tissues taken 8 and 33 days after treatment of rats with DCF, [3H]DCF binding was reduced to 27% and 48% of control levels, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Implicit memory for possible and impossible objects: constraints on the construction of structural descriptions.

Four experiments examined implicit memory or priming effects on an object decision task in which subjects decided whether structurally possible or impossible novel objects could exist in three-dimensional form. Results revealed equivalent levels of priming for possible objects after 1 vs. 4 5-s exposures to the same structural encoding task (Experiment 1) and when objects were studied with a single structural encoding task or 2 different structural encoding tasks (Experiment 3). Explicit memory, by contrast, was greatly affected by both manipulations. However, priming of possible objects was not observed when Ss were given only a single 1-s exposure to perform a structural encoding task (Experiment 2). No evidence for priming of impossible objects was observed in any of the 4 experiments. The data suggest that object decision priming depends on a presemantic structural description system that is distinct from episodic memory.

Implicit memory for unfamiliar objects depends on access to structural descriptions.

We investigated implicit memory for unfamiliar objects with a task in which subjects decided whether structurally possible and impossible line drawings could exist in three-dimensional space. In Experiment 1, significant priming effects on object decision performance were observed after encoding of global, three-dimensional object structure but not local, two-dimensional object features. Explicit memory did not differ significantly as a function of global vs. local study processing. In Experiments 2 and 3, elaborative encoding had different effects on object decision and recognition performance, thus providing evidence for functional dissociation between implicit and explicit memory. Stochastic independence between object decision and recognition performance was also observed. Results were consistent with the hypothesis that implicit memory, as indexed by priming on the object decision task, depends on encoding of and access to structural descriptions of objects.